U.S. patent application number 15/742415 was filed with the patent office on 2018-07-12 for compositions, kits, and methods using interleukin-17c to promote neural growth and/or neural survival.
The applicant listed for this patent is FRED HUTCHINSON CANCER RESEARCH CENTER, UNIVERSITY OF WASHINGTON. Invention is credited to Lawrence Corey, Tao Peng, Jia Zhu.
Application Number | 20180193419 15/742415 |
Document ID | / |
Family ID | 57685748 |
Filed Date | 2018-07-12 |
United States Patent
Application |
20180193419 |
Kind Code |
A1 |
Corey; Lawrence ; et
al. |
July 12, 2018 |
COMPOSITIONS, KITS, AND METHODS USING INTERLEUKIN-17C TO PROMOTE
NEURAL GROWTH AND/OR NEURAL SURVIVAL
Abstract
The present disclosure provides compositions, kits, and methods
of promoting neural growth and/or neural survival using IL-17c. The
compositions, kits, and methods can be used to promote neural
growth and/or neural survival in a variety of conditions where such
growth and survival is beneficial.
Inventors: |
Corey; Lawrence; (Mercer
Island, WA) ; Zhu; Jia; (Kenmore, WA) ; Peng;
Tao; (Kenmore, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FRED HUTCHINSON CANCER RESEARCH CENTER
UNIVERSITY OF WASHINGTON |
Seattle
Seattle |
WA
WA |
US
US |
|
|
Family ID: |
57685748 |
Appl. No.: |
15/742415 |
Filed: |
July 7, 2016 |
PCT Filed: |
July 7, 2016 |
PCT NO: |
PCT/US16/41379 |
371 Date: |
January 5, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62189622 |
Jul 7, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/54 20130101;
A61K 9/7023 20130101; A61P 25/02 20180101; A61K 38/20 20130101;
A61K 9/0014 20130101 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 9/70 20060101 A61K009/70; A61K 9/00 20060101
A61K009/00; A61P 25/02 20060101 A61P025/02 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grants
Al042528, Al030731, Al111780, and Al093746 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of promoting neural growth and/or neural survival in a
subject comprising administering to the subject a therapeutically
effective amount of an IL-17c protein thereby promoting neural
growth and/or neural survival in the subject
2. A method of claim 1, wherein the neural growth is evidenced by
nerve density, neurite growth and/or neurite guidance.
3. A method of claim 1, wherein the IL-17c protein comprises SEQ ID
NO: 1.
4. A method of claim 1, wherein the promoted neural growth and/or
neural survival is found in a sensory or motor neural cell and/or
nerve.
5. A method of claim 4, wherein the administering is in or around a
site of the sensory or motor neural cell and/or nerve.
6. A method of claim 1, wherein the administering is topical.
7. A method of claim 1, wherein the administering is through
application of a transdermal patch.
8. A method of claim 1, wherein the administering is
prophylactic.
9. A method of claim 1, wherein the administering is before an
upcoming insult.
10. A method of claim 9, wherein the upcoming insult is a scheduled
insult.
11. A method of claim 10, wherein the scheduled insult is surgery
or chemotherapy.
12. A method of claim 1 wherein the promoting alleviates a symptom
of neurodegeneration.
13. A method of claim 12 wherein the neurodegeneration is a
peripheral neuropathy.
14. A method of promoting neural growth and/or neural survival
comprising contacting the neural cell or nerve with a
therapeutically effective amount of an IL-17c protein thereby
promoting neural growth and/or neural survival.
15. A method of claim 14, wherein the neural growth and/or neural
survival is evidenced by increased neural cell survival, increased
neurite growth, neurite guidance, and/or increased innervation.
16. A method of claim 14, wherein the IL-17c protein comprises SEQ
ID NO: 1.
17. A method of claim 14, wherein the neural cell or nerve is from
the peripheral nervous system.
18. A method of claim 14, wherein the neural cell or nerve is from
a sensory or motor neural cell or nerve.
19. A method of claim 14, wherein the neural cell or nerve is
within the dermis of a subject.
20. A method of claim 19, wherein the subject is a subject in need
of the promoting neural growth and/or neural survival.
21. A method of claim 20, wherein the promoting alleviates a
symptom of neurodegeneration.
22. A method of claim 21, wherein the neurodegeneration is a
peripheral neuropathy.
23. A composition comprising a therapeutically effective amount of
an IL-17c protein and a pharmaceutically acceptable carrier.
24. A composition of claim 23, wherein the IL-17c protein comprises
SEQ ID NO: 1.
25. A composition of claim 23 wherein the pharmaceutically
acceptable carrier comprises a topical carrier.
26. A composition of claim 24 wherein the pharmaceutically
acceptable carrier is selected from an aqueous carrier, an
oil-based carrier, a fat-based carrier, a fatty alcohol-based
carrier, or a combination thereof.
27. A kit for promoting neural growth and/or neural survival, the
kit comprising a therapeutically effective amount of an IL-17c
protein and instructions for administering the therapeutically
effective amount of the IL-17c protein to a subject.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application No. 62/189,622 filed on Jul. 7, 2015, which is
incorporated herein by reference in its entirety as if fully set
forth herein.
FIELD OF THE DISCLOSURE
[0003] The present disclosure provides compositions, kits, and
methods to promote neural growth and/or neural survival. The use or
stimulation of the cytokine Interleukin-17c (IL-17c) in related
compositions, kits, and methods are described.
BACKGROUND OF THE DISCLOSURE
[0004] There are a variety of conditions where neural growth and/or
neural survival would be beneficial. For example, neurodegeneration
is a damaged state of the nervous system evidenced by injured,
diseased, or dysfunctional neural cells or nerves. Spinal cord
injury and neurodegenerative disorders such as multiple sclerosis
are examples of neurodegeneration. In the peripheral nervous
system, neurodegeneration is referred to as neuropathy.
Neuropathies of the peripheral nervous system are estimated to
affect 20 million people in the United States. Peripheral
neuropathies often cause weakness, paralysis, numbness or pain
(e.g., sensations of burning, stabbing pain, tingling and/or
extreme sensitivity to touch). Approximately 30% of peripheral
neuropathies are caused by diabetes; 30% are idiopathic; and other
causes include autoimmune disorders, tumors, heredity, nutritional
imbalances, infections, chemotherapy, medications, toxins, and
accidents. Promotion of neural growth and/or neural survival would
be beneficial in people and animals suffering from
neurodegeneration.
[0005] Neurotrophic factors are a well-known family of proteins
that support the growth, survival, and maintenance of neurons.
Neurotrophic factors are released by target tissue to guide axonal
growth. Well-known neurotrophic factors include Nerve Growth Factor
(NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3
(NT-3), and Neurotrophin-4 (NT-4).
[0006] Interleukins are a group of cytokines that function as part
of the immune system. The Interleukin-17 family of cytokines is a
group of pro-inflammatory cytokines secreted by activated memory T
cells that play an active role in the inflammatory response. The
Interleukin-17 family of cytokines has been implicated in
inflammatory diseases and autoimmune diseases.
SUMMARY OF THE DISCLOSURE
[0007] The present disclosure provides compositions, kits, and
methods of promoting neural growth and/or neural survival using
IL-17c. The compositions, kits, and methods can be used to promote
neural growth and/or neural survival in a variety of conditions
where such growth and survival is beneficial.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIGS. 1A-1D. Nerve fiber growth during HSV reactivation. 1A.
Nerve fibers in skin biopsies at time of subclinical HSV-2
reactivation (shedding) are stained for NCAM. An increased number
of NCAM+ nerve fibers in the area just below the dermal epidermal
junction, the site of HSV-2 reactivation, is seen as compared to
control biopsy obtained from contralateral genital skin. Cells were
co-stained with an anti-CD8a antibody and DAPI. Scale bar: 50
.mu.m. 1B. Comparison of length and width of NCAM+ nerve fibers
between biopsies obtained at time of HSV-2 shedding and
contralateral control biopsies. Each line represents one individual
(n=4). 1C. The difference of nerve fiber length between biopsies
and contralateral controls at the time of shedding (n=4) versus
biopsies in which no HSV reactivation was detected (n=8). P-value
is derived from 2 samples t-test with unequal variance. 1D. NCAM+
nerve fibers express NGFR and intermediate filaments (peripherin
and NF200). Tissue of 4 weeks post healed asymptomatic shedding
skin biopsies was double stained with anti-NCAM and NGFR,
peripherin or NF200 antibodies. Scale bar: 50 .mu.m.
[0009] FIGS. 2A-2C. Recurrent HSV-2 reactivation induces IL-17c
expression in keratinocytes. 2A. Isolation of keratinocytes above
basement membrane by laser capture microdissection (LCM). 2B. HSV
infection in keratinocytes induced IL-17c expression in vivo.
Comparison of expression of cytokines/chemokines (top panel) and
six different members of IL-17 (bottom panel) in keratinocytes
isolated from lesion and post healed skin (Asymptomatic shedding)
biopsies to those from contralateral control biopsies. 2C. IL-17c
protein expression in epidermal keratinocytes in skin biopsies
during lesion and shedding (clinical quiescence 8 weeks shedding).
IL-17c expression was detected by immunofluorescent staining with
an anti-IL-17c antibody and nuclei stained with DAPI. Scale bar: 50
.mu.m.
[0010] FIG. 3. Expression of keratin 5 (KRT5) and 14 (KRT14) in
laser captured keratinocytes (Kera), CD8a+CD8 T cells (CD8) and
CD1a+ Langerhans cells (CD1) from control (ctrl) and post healed
(PH) genital skin biopsies during recurrent HSV-2 infection.
Y-axis: intensity values from normalized Illumina BeadArray data.
The displayed values are the averages for keratinocytes (n=4),
CD8a+CD8 T cells (n=8) and CD1a+ Langerhans cells (n=8).
[0011] FIGS. 4A-4F. Peripheral nervous system expression of
IL-17RE, the orphan receptor for IL-17c. 4A. Nerve fibers in
genital skin expressed IL-17RE. In skin biopsies obtained during
recurrent HSV-2 infection, IL-17RE cells exhibited elongated fiber
like shapes and were distinct from CD15+, CD8a+ and CD4+ cells.
Double immunofluorescent staining with anti-IL-17RE and anti-CD15,
CD8a or CD4 antibodies revealed no co-staining. Scale=50 .mu.m. 4B.
Double immunofluorescent staining with anti-NCAM and anti-IL-17RE
antibodies in lesion biopsies. Nuclei stained with DAPI. Scale bar:
50 .mu.m. 4C. Double immunofluorescent staining with
anti-peripherin and anti-IL-17RE antibodies revealed expression of
IL-17RE on peripherin+ nerve endings in genital 4 weeks post healed
skin biopsies in epidermis (left) and dermis (right). Nuclei
stained with DAPI. Scale bar=50 .mu.m. 4D. Single immunofluorescent
staining with anti-IL-17RE in sensory neurons from human fetal DRG
showed staining in both neuronal cell bodies and nerve fibers.
Insets show enlarged pictures of IL-17RE expression in cell bodies
(top) and axons (bottom). Scale bar=500 .mu.m. 4E. Detection of
IL-17RE RNA expression in sensory neurons in human fetal DRGs using
FISH. TUBB3: tubulin beta 3 class III. Three representative images
are displayed. Scale bar=50 .mu.m. 4F. IL-17RE expression in a
subset of NF200+(left panel) or peripherin+ neurons (right panel)
and axons in human fetal DRG. Scale bar: 50 .mu.m.
[0012] FIGS. 5A-5D. IL-17c expression in HSV infected human primary
keratinocytes. 5A. IL-17c RNA expression in a time course of HSV
infected keratinocytes. Cells were mock infected or infected with
HSV-2 (HG52) in the absence or presence of acyclovir (30 .mu.M) or
with UV inactivated viruses (left panel) or such cells were
infected with HSV-1 (KOS) and three HSV-1 mutants with deletion in
ICP0, ICP22 and ICP8, respectively (right panel). Y-axis is fold
change of RNA levels above mock infected cells; x-axis is time in
hours. Gene expression for IL-17c was assayed by quantitative PCR.
5B. IL-17c protein expression in HSV-2 infected keratinocytes.
Cells mock (bottom) or HSV-2 infected (top) for 7 hours at MOI of 1
and 10 were analyzed for IL-17c expression by immunofluorescent
staining with an anti-IL-17c antibody. Nuclei stained with DAPI.
Graph is quantification of staining as the percentage of IL-17c
expressing cells. Error bars represent one standard deviation from
the mean of three replicates. 5C. HSV infection and bacterial TLR
agonists independently induce IL-17c expression in primary
keratinocytes. A TLR2 neutralizing antibody was added to the cells
one hour before infection or peptidoglycan (PGN) treatment. For
combination of HSV infection and PGN treatment, cells were infected
with KOS at MOI of 1 for 3 hours and were then treated with PGN (2
ug/mL) for an additional 3 hours (left panel). For combination of
HSV infection and flagellin treatment, cells were mock infected or
infected with ICP8mu for 7 hours, or untreated or treated with
flagellin (100 ng/ml) for 1 hour or infected with ICP8mu for 6
hours and then treated with flagellin for 1 hour (right panel).
Error bars represent one standard deviation from the mean of three
biological replicates. Gene expression for IL-17c was assayed by
quantitative PCR. 5D. NF-.kappa.B and IRF-3 mediated IL-17c
induction during HSV infection of human primary keratinocytes.
Cells were transfected with control siRNA (siRNA_ctrl) or siRNA for
NF-.kappa.B, IRF1, IRF-3 or IRF7 for 48 hours and then mock
infected or infected with KOS for 3 and 6 hours. Gene expression
for IL-17c was assayed by quantitative PCR. Error bars represent
one standard deviation from the mean of three biological
replicates.
[0013] FIGS. 6A and 6B. siRNA knock-down of NFKB1, IRF1, IRF3 and
IRF7 and similar expression of IL-17c in HSV infected primary human
keratinocytes with reduced expression of IFI16 and PML. 6A. siRNA
knock-down of gene expression of NF-.kappa.B, IRF1, IRF3 or IRF7 in
primary keratinocytes. Primary keratinocytes were transfected with
control siRNA or siRNA for NF-.kappa.B, IRF1, IRF3 or IRF7 for 48
hours and then mock infected or infected with KOS for 3 and 6
hours. Gene expression for NF-.kappa.B, IRF1, IRF3 and IRF7 were
assayed by quantitative PCR. 6B. Expression of IL-17c in HSV
infected keratinocytes with reduced expression of IFI16 and PML.
Primary keratinocytes were transfected with control siRNA or siRNA
for IFI16 or PML for 48 hours and then mock infected or infected
with KOS for 6 hours.
[0014] FIGS. 7A-7C. Blocking IL-17c signaling does not have
significant effect on HSV gene expression or viral titers in
infected human primary keratinocytes. Cells were untreated or
pre-treated with an IL-17RA neutralizing antibody or matching
control IgG for one hour before HSV-1 infection (7A & 7B). Gene
expression of ICP27 was assayed by quantitative PCR and viral
titers were determined by plaque assay in Vero cells. 7C. Primary
keratinocytes were transfected with control siRNA or siRNA for
IL-17RE for 48 hours and then mock infected or infected with KOS
for 3 and 6 hours. Expression of ICP27, gB and IL-17RE was
determined by quantitative PCR and viral titers were determined by
plaque assay in Vero cells.
[0015] FIGS. 8A-8D. IL-17c stimulated neurite growth of
differentiated SY5Y neurons. 8A. IL-17c induced directional neurite
growth of differentiated SY5Y cells in a microfluidic device. SY5Y
cells were differentiated with all trans retinoid acids (ATRA) at
20 .mu.g/mL for 4 days and then placed in the wells on the left
side of a microfluidic device with culture medium alone (M) or
medium plus IL-17c or NGF on the other side. After 10 days of
culture cells were fixed and stained with a PGP9.5 antibody. 8B-8D.
IL-17c induced significantly more and longer neurites of
differentiated SY5Y cells as compared to medium only or medium plus
NGF. 8B. Comparison of growth cones of two neurites from medium
only and medium plus IL-17c devices. 8C. The length of individual
neurites that were extended into the main channel on the right side
was measured in IMAGEJ. 8D. The error bar represents one standard
deviation of data (number of neurites and total neurite length)
from 3 microfluidic devices for each condition.
[0016] FIGS. 9A-9H. IL-17c induced neurite growth and branch points
in HSN. HSN were isolated from individual human fetal spinal tissue
and cultured in full neural medium only or medium plus IL-17c or
NGF. 9A. Images of cultured HSN in the presence of IL-17c at 75
(top) and 90 (bottom) hours after plating. 9B. Live imaging of HSN
to measure neurite length (left graph), neurite branch points
(middle graph) and cell body area (right graph) every hour for 16
hours from hours 75 to 90 after HSN were plated in culture medium
or medium plus IL-17c or NGF. 9C. Growth rates of neurite length
(left graph), neurite branch points (middle graph) and cell body
area (right graph) of cultured HSN from hours 75 to 90 after HSN
were plated. 9D. A microfluidic device with three channels. HSN
were placed in the middle channel and medium only (M) and medium
plus IL-17c was placed on the left and right channels,
respectively. DRG=dorsal root ganglia. 9E & 9F. HSN extended
significantly longer neurites with more branch points into the
channel with IL-17c containing medium. HSN were fixed and stained
with PGP9.5 after 16 days of culture (9E) and number of neurites,
total length and branch points were counted (9F). Scale bar=500
.mu.m. 9G & 9H. The HSN neurites expressed IL-17RE. HSN in the
three channel device were double stained with PGP9.5 and IL-17RE
antibodies (9G). Comparison of growth cones of neurites from medium
only and IL-17c containing channels (9H).
[0017] FIGS. 10A-10C. Bright field images of cultured human fetal
sensory neurons (left) at 75 (top) and 90 hours (bottom); and cell
body and neurite length and branch points were measured with the
Incucyte neuro-track image analysis software module for both time
points (right). FIG. 10A; medium. FIG. 10B; NGF. FIG. 100;
IL-17c.
[0018] FIGS. 11A-11D. IL-17c protects mouse primary cortical
neurons and human primary keratinocytes from apoptosis during HSV
infection. 11A. HSV infection induces expression of IL-17c and
IL-17RE in MCN. Cells were infected with HSV-1 (K26) at MOI of 5
for 6, 12, 24 and 36 hours. Y-axis is fold change over mock
infected MCN. Gene expression was determined by quantitative PCR.
11B. Detection by immunofluorescence of cleaved caspase 3 levels in
K26 infected MCN. MCN were untreated or pre-treated with murine
IL-17c (mIL17c) for 24 hours in the presence of a murine IL-17RA
neutralizing antibody (anti-mIL17RA) or matching control rat IgG
before K26 infection at MOI of 5 for 16 hours. Cells were stained
with DAPI for cell nucleus and an antibody for cleaved caspase 3.
K26 infected cells express GFP. 11C. Percentages of cleaved caspase
3+ neurons in K26 infected neurons pretreated with mIL-17c in the
presence of anti-mIL17RA or control IgG. Error bars represent one
standard deviation from the mean of three replicates. 11D.
Exogenous human IL-17c (hIL17c) treatment provides a survival
signal to keratinocytes (Kera) during HSV infection and a human
IL-17RA neutralizing antibody (anti-hIL17RA) blocks its effect.
Keratinocytes were untreated or pre-treated with hIL-17c for 12
hours in the presence of anti-hIL17RA or control IgG and then were
mock or K26 infected (MOI of 2) for 12 hours.
[0019] FIG. 12. Blocking IL-17c signaling does not have significant
effect on HSV gene expression in infected mouse primary neurons.
Cells were untreated or pre-treated with an IL-17RA neutralizing
antibody or matching control IgG for one hour before HSV-1
Infection. ICP27 expression was determined by quantitative PCR.
DETAILED DESCRIPTION
[0020] There are a variety of conditions where neural growth and/or
neural survival would be beneficial. For example, neurodegeneration
is a damaged state of the nervous system evidenced by injured,
diseased, or dysfunctional neural cells or nerves. Spinal cord
injury and neurodegenerative disorders such as multiple sclerosis
are examples of neurodegeneration. In the peripheral nervous
system, neurodegeneration is referred to as neuropathy. Thus,
neuropathies are a subtype of neurodegeneration. Neuropathies of
the peripheral nervous system are estimated to affect 20 million
people in the United States. Peripheral neuropathies often cause
weakness, paralysis, numbness or pain (e.g., sensations of burning,
stabbing pain, tingling and/or extreme sensitivity to touch).
Approximately 30% of peripheral neuropathies are caused by
diabetes; 30% are idiopathic; and other causes include autoimmune
disorders, tumors, heredity, nutritional imbalances, infections,
chemotherapy, medications, toxins, and accidents. Promotion of
neural growth and/or neural survival would be beneficial in people
and animals suffering from neurodegeneration.
[0021] Interleukins are a group of cytokines that function as part
of the immune system. The Interleukin-17 family of cytokines is a
group of pro-inflammatory cytokines secreted by activated memory T
cells that play an active role in the inflammatory response. The
Interleukin-17 family of cytokines has been implicated in
inflammatory diseases and autoimmune diseases.
[0022] The present disclosure provides compositions, kits, and
methods of promoting neural growth and/or neural survival using
IL-17c. The compositions, kits, and methods can be used to promote
neural growth and/or neural survival in a variety of conditions
where such growth and survival is beneficial.
[0023] The Interleukin-17 cytokine family has six cytokines
(Interleukins-17A through -17F); and there are five receptors
(Interleukin-17RA through -17RE). IL-17 regulates the innate immune
function of epithelial cells. Interleukin-17c (IL-17c) (SEQ ID NO.
1) is expressed in a wide variety of tissues. IL-17c is a 40 kDa
protein having 197 amino acids, and 23% amino acid sequence
identity to IL-17A. IL-17c binds to IL-17RE, a member of the IL-17
receptor family, and signals through a receptor heterodimeric
complex formed by IL-17RA and IL-17RE. For more information
regarding the interleukin-17 cytokine family, see, e.g., Krstic et
al., Protein Pept Lett. 2015; 22(7):570-8; Shabgah et al., Postepy
Dermatol Alergol. 2014 August; 31(4):256-61; Gaffen, Curr Opin
Immunol. 2011 October; 23(5):613-9; Gaffen et al., Vitam Horm.
2006; 74:255-82; and Moseley et al., Cytokine Growth Factor Rev.
2003 April; 14(2):155-74.
[0024] IL-17c proteins include SEQ ID NO: 1 and biologically active
analogues thereof. Biologically active analogues include proteins
having at least 70% sequence identity with SEQ ID NO:1; at least
75% sequence identity with SEQ ID NO:1; at least 80% sequence
identity with SEQ ID NO:1; at least 81% sequence identity with SEQ
ID NO:1; at least 82% sequence identity with SEQ ID NO:1; at least
83% sequence identity with SEQ ID NO:1; at least 84% sequence
identity with SEQ ID NO:1; at least 85% sequence identity with SEQ
ID NO:1; at least 86% sequence identity with SEQ ID NO:1; at least
87% sequence identity with SEQ ID NO:1; at least 88% sequence
identity with SEQ ID NO:1; at least 89% sequence identity with SEQ
ID NO:1; at least 90% sequence identity with SEQ ID NO:1; at least
91% sequence identity with SEQ ID NO:1; at least 92% sequence
identity with SEQ ID NO:1; at least 93% sequence identity with SEQ
ID NO:1; at least 94% sequence identity with SEQ ID NO:1; at least
95% sequence identity with SEQ ID NO:1; at least 96% sequence
identity with SEQ ID NO:1; at least 97% sequence identity with SEQ
ID NO:1; at least 98% sequence identity with SEQ ID NO:1; or at
least 99% sequence identity with SEQ ID NO:1; and the biologically
active analogue also has at least 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, 100%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 600%,
700%, 800%, 900%, 1000% or more of the biological activity of the
IL-17c protein of SEQ ID NO:1.
[0025] "% sequence identity" refers to a relationship between two
or more sequences, as determined by comparing the sequences. In the
art, "identity" also means the degree of sequence relatedness
between sequences as determined by the match between strings of
such sequences. "Identity" (often referred to as "similarity") can
be readily calculated by known methods, including those described
in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford
University Press, NY (1988); Biocomputing: Informatics and Genome
Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer
Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular
Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence
Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford
University Press, NY (1992). Preferred methods to determine
sequence identity are designed to give the best match between the
sequences tested. Methods to determine sequence identity and
similarity are codified in publicly available computer programs.
Sequence alignments and percent identity calculations may be
performed using the Megalign program of the LASERGENE
bioinformatics computing suite (DNASTAR, Inc., Madison, Wis.).
Multiple alignment of the sequences can also be performed using the
Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153
(1989) with default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Relevant programs also include the GCG suite of
programs (Wisconsin Package Version 9.0, Genetics Computer Group
(GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul, et al., J.
Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison,
Wis.); and the FASTA program incorporating the Smith-Waterman
algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.]
(1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor.
Publisher: Plenum, New York, N.Y. Within the context of this
disclosure it will be understood that where sequence analysis
software is used for analysis, the results of the analysis are
based on the "default values" of the program referenced. "Default
values" mean any set of values or parameters which originally load
with the software when first initialized.
[0026] The biological activity of IL-17c and biologically active
analogues thereof can be assessed using any relevant activity
assay. In particular embodiments, activity can be assessed by
treating human fetal sensory neurons with SEQ ID NO: 1 and/or
biologically active analogues thereof. Neurite length, density,
and/or branching points can be assessed within 3-4 days of
treatment with IL-17c and/or biologically active analogues and/or
other relevant control compounds.
[0027] Biologically active analogues of IL-17c can include
variants, D-substituted analogs and modifications thereof.
[0028] "Variants" of proteins disclosed herein include proteins
having one or more amino acid additions, deletions, stop positions,
or substitutions, as compared to SEQ ID NO: 1.
[0029] An amino acid substitution can be a conservative or a
non-conservative substitution. Variants of proteins disclosed
herein can include those having one or more conservative amino acid
substitutions. A "conservative substitution" involves a
substitution found in one of the following conservative
substitutions groups: Group 1: alanine (Ala or A), glycine (Gly or
G), Ser, Thr; Group 2: aspartic acid (Asp or D), Glu; Group 3:
asparagine (Asn or N), glutamine (Gln or Q); Group 4: Arg, lysine
(Lys or K), histidine (His or H); Group 5: Ile, leucine (Leu or L),
methionine (Met or M), valine (Val or V); and Group 6: Phe, Tyr,
Trp.
[0030] Additionally, amino acids can be grouped into conservative
substitution groups by similar function, chemical structure, or
composition (e.g., acidic, basic, aliphatic, aromatic,
sulfur-containing). For example, an aliphatic grouping may include,
for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other
groups containing amino acids that are considered conservative
substitutions for one another include: sulfur-containing: Met and
Cys; acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or
slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar,
negatively charged residues and their amides: Asp, Asn, Glu, and
Gln; polar, positively charged residues: His, Arg, and Lys; large
aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and
large aromatic residues: Phe, Tyr, and Trp. Additional information
is found in Creighton (1984) Proteins, W.H. Freeman and
Company.
[0031] "D-substituted analogs" include SEQ ID NO: 1 having one or
more L-amino acids substituted with one or more D-amino acids. The
D-amino acid can be the same amino acid type as that found in SEQ
ID NO: 1 or can be a different amino acid. Accordingly,
D-substituted analogs can also be variants.
[0032] Modified IL-17c (modifications) include SEQ ID NO: 1 changed
to have a beneficial property such as (a) increased protein serum
half-life and/or functional in vivo half-life, (b) reduced protein
antigenicity, (c) increased protein storage stability, (d)
increased protein solubility, (e) increased bioavailability (e.g.
increased area under the curve (AUC)); (f) increased
bioaccessibility to selected areas (e.g., to cross the blood brain
barrier or to reach other physiologically protected areas); and/or
(g) targeted delivery to reduce required dosage and/or avoid
off-target side effects.
[0033] In particular embodiments, modified IL-17c proteins include
IL-17c wherein one or more amino acids have been replaced with a
non-amino acid component, or where the amino acid has been
conjugated to a functional group or a functional group has been
otherwise associated with an amino acid. The modified amino acid
may be, for example, a glycosylated amino acid, a PEGylated amino
acid, a farnesylated amino acid, an acetylated amino acid, a
biotinylated amino acid, an amino acid conjugated to a lipid
moiety, or an amino acid conjugated to an organic derivatizing
agent. Amino acid(s) can be modified, for example,
co-translationally or post-translationally during recombinant
production (e.g., N-linked glycosylation at N-X-S/T motifs during
expression in mammalian cells) or modified by synthetic means. The
modified amino acid can be within the sequence or at the terminal
end of a sequence. Modifications also include nitrited IL-17c
protein.
[0034] Regarding PEGylated amino acids, covalent attachment of
proteins to PEG has proven to be a useful method to increase the
circulating half-lives of proteins in the body (Abuchowski, A. et
al., Cancer Biochem. Biophys., 1984, 7:175-186; Hershfield, M. S.
et al., N. Engl. J. Medicine 316:589-596; and Meyers, F. J. et al.,
Clin. Pharmacol. Ther., 1991, 49:307-313). The attachment of PEG to
proteins not only protects the molecules against enzymatic
degradation, but also reduces their clearance rate from the body.
The size of PEG attached to a protein has significant impact on the
circulating half-life of the protein. The ability of PEGylation to
decrease clearance is generally not a function of how many PEG
groups are attached to the protein, but the overall molecular
weight of the altered protein. PEGylation decreases the rate of
clearance from the bloodstream by increasing the apparent molecular
weight of the molecule. Up to a certain size, the rate of
glomerular filtration of proteins is inversely proportional to the
size of the protein. Usually the larger the PEG is, the longer the
in vivo half-life of the attached protein is. In addition,
PEGylation can also decrease protein aggregation (Suzuki et al.,
Biochem. Bioph. Acta vol. 788, pg. 248 (1984)), alter protein
immunogenicity (Abuchowski et al.; J. Biol. Chem. vol. 252 pg. 3582
(1977)), and increase protein solubility as described, for example,
in PCT Publication No. WO 92/16221. Several sizes of PEGs are
commercially available (Nektar Advanced PEGylation Catalog
2005-2006; and NOF DDS Catalogue Ver 7.1). A variety of active PEGs
have been used including mPEG succinimidyl succinate, mPEG
succinimidyl carbonate, and PEG aldehydes, such as
mPEG-propionaldehyde.
[0035] Several methods of PEGylating proteins have been reported in
the literature. For example, N-hydroxy succinimide (NHS)-PEG was
used to PEGylate the free amine groups of lysine residues and
N-terminus of proteins; PEGs bearing aldehyde groups have been used
to PEGylate the amino-termini of proteins in the presence of a
reducing reagent; PEGs with maleimide functional groups have been
used for selectively PEGylating the free thiol groups of cysteine
residues in proteins; and site-specific PEGylation of
acetyl-phenylalanine residues can be performed.
[0036] While exemplary sequences are provided herein, sequence
information provided by public databases can be used to identify
related and relevant protein sequences and associated nucleic acid
sequences encoding such proteins.
[0037] IL-17c (including biologically active analogues thereof)
(individually and collectively, "active ingredients") can be
provided alone or in combination within a composition. In
particular embodiments, a composition includes at least one active
ingredient and at least one pharmaceutically acceptable carrier. In
addition or alternatively to administering an active ingredient
directly as a therapeutic, compounds that stimulate IL-17c can also
be administered. Such compounds include, for example, HSV (e.g.,
inactivated HSV) and toll-like receptor (TLR) ligands. These
compounds, as well as their biologically active analogues, are also
active ingredients within the scope of the disclosure.
[0038] Inactivated HSV is HSV in a non-infective (inactive) form.
Examples of virus inactivation methods include solvent and/or
detergent inactivation, pasteurization (e.g., heating to high
temperatures), pH inactivation (e.g., using an acidic or alkaline
pH; see, e.g., Lancz & Sample, Archives of Virology, March
1985, 84(1), 141-146 describing the thermal sensitivity of HSV at
an alkaline pH), and irradiation (e.g., ultraviolet (UV) or gamma
irradiation). HSV can be inactivated and safely administered to
subjects. See, e.g., Whitley, Herpes Simplex Viruses, p. 2461-2509.
In D. M. Knipe and P. M. Howley (ed.), Fields Virology, Fourth ed,
vol. 2. Lippincott Williams & Wilkins, Philadelphia (2001).
[0039] Toll like receptors (TLRs) are a family of pattern
recognition receptors that are activated by specific components of
microbes and certain host molecules. They constitute the first line
of defense against many pathogens and play a crucial role in the
function of the innate immune system. It is estimated that most
mammalian species have between ten and fifteen types of Toll-like
receptors.
[0040] TLR ligands are widely available commercially, for example
from Apotech and InvivoGen. Examples of TLR2 ligands include fungi,
lipoglycans, lipopolysaccharides, lipoproteins, lipoteichoic acids,
peptidoglycans, viral glycoproteins, and zymosan. More particular
examples available from Invivogen include heat-killed: Acholeplasma
laidlawii (Mycoplasma); Escherichia coli; Helicobacter pylori;
Listeria monocytogenes; Legionella pneumophila; Lactobacillus
rhamnosus; Mycoplasma fermentans; Mycobacterium tuberculosis;
Pseudomonas aeruginosa; Porphyromonas gingivalis; Staphylococcus
aureus; Staphylococcus epidermidis; Streptococcus pneumonia; and
Salmonella typhimurium.
[0041] An example of a TLR5 ligand includes flagellin. More
particular examples available from InvivoGen include flagellin
from: Bacillus subtilis; Pseudomonas aeruginosa; and Salmonella
typhimurium; including recombinant and mutant forms.
[0042] For additional information on TLRs and TLR ligands, see,
Akira, Curr Opin Immunol 2003; 15(1): 5-11 and Akira and Hemmi,
Immunol Lett 2003; 85(2): 85-95.
[0043] Salts and/or pro-drugs of active ingredients can also be
used.
[0044] A pharmaceutically acceptable salt includes any salt that
retains the activity of the active ingredient and is acceptable for
pharmaceutical use. A pharmaceutically acceptable salt also refers
to any salt which may form in vivo as a result of administration of
an acid, another salt, or a prodrug which is converted into an acid
or salt.
[0045] A prodrug includes an active ingredient which is converted
to a therapeutically active compound after administration, such as
by cleavage of a protein or by hydrolysis of a biologically labile
group.
[0046] In some embodiments, the compositions include active
ingredients of at least 0.1% weight/volume (w/v) or weight/weight
(w/w) of the composition; at least 1% w/v or w/w of composition; at
least 10% w/v or w/w of composition; at least 20% w/v or w/w of
composition; at least 30% w/v or w/w of composition; at least 40%
w/v or w/w of composition; at least 50% w/v or w/w of composition;
at least 60% w/v or w/w of composition; at least 70% w/v or w/w of
composition; at least 80% w/v or w/w of composition; at least 90%
w/v or w/w of composition; at least 95% w/v or w/w of composition;
or at least 99% w/v or w/w of composition.
[0047] Exemplary pharmaceutically acceptable carriers include any
and all absorption delaying agents, antioxidants, binders,
buffering agents, bulking agents or fillers, chelating agents,
coatings, disintegration agents, dispersion media, gels, isotonic
agents, lubricants, preservatives, release modifiers, salts,
solvents or co-solvents, stabilizers, surfactants, and delivery
vehicles.
[0048] Exemplary antioxidants include ascorbic acid, methionine,
and vitamin E.
[0049] Exemplary buffering agents include citrate buffers,
succinate buffers, tartrate buffers, fumarate buffers, gluconate
buffers, oxalate buffers, lactate buffers, acetate buffers,
phosphate buffers, histidine buffers, and trimethylamine salts.
[0050] An exemplary chelating agent is EDTA.
[0051] Exemplary isotonic agents include polyhydric sugar alcohols
including trihydric and higher sugar alcohols, such as glycerin,
erythritol, arabitol, xylitol, sorbitol, and mannitol.
[0052] Exemplary preservatives include phenol, benzyl alcohol,
meta-cresol, methyl paraben, propyl paraben,
octadecyldimethylbenzyl ammonium chloride, benzalkonium halides,
hexamethonium chloride, alkyl parabens such as methyl and propyl
paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.
[0053] Exemplary release modifiers can include surfactants,
detergents, internal phase viscosity enhancers, complexing agents,
surface active molecules, co-solvents, chelators, stabilizers,
derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC),
HPMC acetate, cellulose acetate, pluronics (e.g., F68/F127),
polysorbates, Span.RTM. (Croda Americas, Wilmington, Del.),
poly(vinyl alcohol) (PVA), Brij.RTM. (Croda Americas, Wilmington,
Del.), sucrose acetate isobutyrate (SAIB), salts, and buffers.
[0054] Acid addition salts can be prepared from an inorganic acid
or an organic acid. Examples of such inorganic acids are
hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric
and phosphoric acid. Appropriate organic acids can be selected from
aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic,
carboxylic and sulfonic classes of organic acids.
[0055] Base addition salts include metallic salts made from
aluminum, calcium, lithium, magnesium, potassium, sodium and zinc
or organic salts made from N,N'-dibenzylethylene-diamine,
chloroprocaine, choline, diethanolamine, ethylenediamine,
N-methylglucamine, lysine, arginine and procaine.
[0056] Useful solvents include water, ethanol, dimethyl sulfoxide
(DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl
alcohol (IPA), ethyl benzoate, and benzyl benzoate.
[0057] Stabilizers refer to a broad category of excipients which
can range in function from a bulking agent to an additive which
solubilizes the active ingredient or helps to prevent denaturation
or adherence to the container wall. Typical stabilizers can include
polyhydric sugar alcohols; amino acids, such as arginine, lysine,
glycine, glutamine, asparagine, histidine, alanine, ornithine,
L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic
sugars and sugar alcohols, such as lactose, trehalose, stachyose,
mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol,
glycerol, and cyclitols, such as inositol; PEG; amino acid
polymers; sulfur-containing reducing agents, such as urea,
glutathione, thioctic acid, sodium thioglycolate, thioglycerol,
alpha-monothioglycerol, and sodium thiosulfate; low molecular
weight polypeptides (i.e., <10 residues); proteins such as human
serum albumin, bovine serum albumin, gelatin or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides
such as xylose, mannose, fructose and glucose; disaccharides such
as lactose, maltose and sucrose; trisaccharides such as raffinose,
and polysaccharides such as dextran. Stabilizers are typically
present in the range of from 0.1 to 10,000 parts by weight based on
active ingredient weight.
[0058] The compositions disclosed herein can be formulated for
administration by, for example, injection, inhalation, infusion,
perfusion, lavage, ingestion, or absorption. The compositions
disclosed herein can further be formulated for transdermal,
intravenous, intradermal, intracranial, intracerebroventricular
(ICV), intranasal, intraarterial, intranodal, intralymphatic,
intraperitoneal, intralesional, intraprostatic, intravaginal,
intrarectal, topical, intrathecal, intratumoral, intramuscular,
intravesicular, oral and/or subcutaneous administration.
Compositions may be formulated for administration by
sustained-release systems or by implantation devices. In certain
embodiments, the compositions may be administered by bolus
injection or continuously by infusion. Compositions may be
administered by local administration or systemic
administration.
[0059] For injection, compositions can be formulated as aqueous
solutions, such as in buffers including Hanks' solution, Ringer's
solution, or physiological saline. The aqueous solutions can
contain formulatory agents such as suspending, stabilizing, and/or
dispersing agents. Alternatively, the formulation can be in
lyophilized and/or powder form for constitution with a suitable
vehicle, e.g., sterile pyrogen-free water, before use.
[0060] For oral administration, the compositions can be formulated
as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries, suspensions and the like. For oral solid formulations
such as, for example, powders, capsules and tablets, suitable
excipients include binders (gum tragacanth, acacia, cornstarch,
gelatin), fillers such as sugars, e.g. lactose, sucrose, mannitol
and sorbitol; dicalcium phosphate, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate; cellulose
preparations such as maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose,
and/or polyvinylpyrrolidone (PVP); granulating agents; and binding
agents. If desired, disintegrating agents can be added, such as
corn starch, potato starch, alginic acid, cross-linked
polyvinylpyrrolidone, agar, and alginic acid or a salt thereof such
as sodium alginate. If desired, solid dosage forms can be
sugar-coated or enteric-coated using standard techniques. Flavoring
agents, such as peppermint, oil of wintergreen, cherry flavoring,
orange flavoring, etc. can also be used.
[0061] Compositions can be formulated for topical administration.
Topical administration refers to administration of a composition at
the point of application. Topically applying describes application
onto one or more surfaces including epithelial surfaces. A
substance delivered by topical administration may not reside in the
skin for an extended period of time, but instead may penetrate into
localized tissue, deep tissue and/or synovial fluids in order to
have an effect on localized tissue, deep tissue, or joints, or any
combination thereof.
[0062] Compositions can be formulated for transdermal delivery.
Transdermal delivery refers to the delivery of a compound, for
example, an active ingredient of this disclosure or other
therapeutic agent, through one or more layers of the skin (e.g.,
epidermis and dermis). Transdermal delivery may include
administration of the composition to the skin surface of a subject
so that the active ingredient passes through the skin tissue and
into deeper tissue thereby providing effects in deep tissue. In
some embodiments, transdermal delivery systems include use of a
patch, iontophoresis, or magnetophoresis, or any combination
thereof. In some embodiments, transdermal delivery is enhanced,
wherein enhancement may be through chemical or physical means.
[0063] A patch refers to a medicated patch, e.g., a patch with a
composition including at least one active ingredient that is placed
on the skin to deliver a dosage of the active ingredient through
the skin and into the surrounding tissue. In some embodiments, the
active ingredient may penetrate deeply below the skin to a site for
deep tissue effects. In some embodiments, the active ingredient
penetrates just below the skin to a localized site for a local
effect. In some embodiments, the dosage of the active ingredient
provides minimal entry of the active ingredient into the blood
stream. In other embodiments, the dosage provides no entry of the
active ingredient into the blood stream.
[0064] Transdermal patches are a well-accepted technology used to
deliver a wide variety of active ingredients. Patches may be placed
on the skin for specified therapeutic time periods. Patches may
include an adhesive to remain in place when placed on the skin or
may be adhered by other means including adhesive tape or strips. In
addition, patches may be perforated or stretchable in order that
they may be wrapped around an appendage or body part. In certain
embodiments, a stretchable patch may be wrapped fully around an
appendage or body part. In alternative embodiments, a stretchable
patch may be wrapped partly around an appendage or body part. For
example, a patch may be wrapped around a knee, ankle, leg, elbow,
wrist, finger, arm, or neck.
[0065] Conventional dermal patches include a carrier that holds an
active ingredient and allows the active ingredient to be released
onto a subject's skin for absorption. Many different kinds of
dermal patches are known, including matrix-type patches,
reservoir-type patches, multi-laminate drug-in-adhesive type
patches, monolithic drug-in-adhesive type patches, and many others.
Such patches can be readily prepared using technology which is
known in the art such as described in Remington's Pharmaceutical
Sciences, 18th or 19th editions, published by the Mack Publishing
Company of Easton, Pa. and "Transdermal And Topical Drug Delivery
Systems" (Tapash K. Ghosh et al. eds., 1997); see also Kristine
Knutson and Lynn K. Pershing, Topical Drugs, in Remington: The
Science And Practice Of Pharmacy 866-885 (Alfonso R. Gennaro ed.,
1995).
[0066] A penetration enhancer may be used with the compositions.
Penetration enhancers refer to agents known to accelerate the
delivery of an active ingredient through the skin. Suitable
penetration enhancers include dimethylsulfoxide (DMSO), dimethyl
formamide (DMF), allantoin, urazole, N,N-dimethylacetamide (DMA),
decylmethylsulfoxide (C.sub.10 MSO), polyethylene glycol
monolaurate (PEGML), propylene glycol (PG), propylene glycol
monolaurate (PGML), glycerol monolaurate (GML), lecithin, the
1-substituted azacycloheptan-2-ones, particularly
1-n-dodecylcyclazacycloheptan-2-one (available under the trademark
Azone.RTM. from Whitby Research Incorporated, Richmond, Va.),
alcohols including menthol, and the like. The permeation enhancer
may also be a vegetable oil. Such oils include safflower oil,
cottonseed oil and corn oil. Additional penetration enhancers may
generally be found in Remington's Pharmaceutical Sciences, 18th or
19th editions, published by the Mack Publishing Company of Easton,
Pa. In certain embodiments, the permeation enhancer is a component
of the composition. In one embodiment, a patch includes a
permeation enhancer in an amount effective to enhance promotion of
neural growth and/or neural survival by an active ingredient. In
some embodiments physical permeation enhancer techniques may be
used including magnetophoresis, iontophoresis or a battery-powered
electronic stimulant.
[0067] Iontophoresis can be used for transdermal active ingredient
delivery. Iontophoresis, also known as Electromotive Drug
Administration (EMDA), is a technique using a small electric charge
to deliver an active ingredient or other chemical through the skin.
It may function similar to an injection without the needle, for
example EMDA may be used for localized entry of an active
ingredient into the skin. In addition, EMDA may be used for
concentrated application of an active ingredient under the
skin.
[0068] Magnetophoresis refers to the motion of dispersed magnetic
particles relative to a fluid under the influence of a magnetic
field. Magnetophoresis may provide enhancing delivery across
biological barriers, including intact skin. In some embodiments,
iontophoresis or magnetophoresis may be used as a transdermal
delivery system alone or in combination with other forms of
administration.
[0069] Microneedle technology may be used. Microneedle transdermal
delivery systems include microneedle patches as well as microneedle
systems that can accommodate transdermal delivery of larger volumes
of active ingredient. Microneedles may be solid or hollow, and
allow for delivery of small molecule, large molecule and
biologically active ingredients. Microneedle devices are
well-suited for dermal skin targets, and are available in a variety
of lengths, depending on the desired depth of the delivery. Hollow
microneedles are available in a variety of sizes to accommodate
various volumes of active ingredient. Dissolving microneedle
patches may be also be used.
[0070] Compositions can also be formulated as depot preparations.
Depot preparations can be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salts.
[0071] Additionally, compositions can be formulated as
sustained-release systems utilizing semipermeable matrices of solid
polymers containing at least one active ingredient. Various
sustained-release materials have been established and are well
known by those of ordinary skill in the art. Sustained-release
systems may, depending on their chemical nature, release active
ingredients following administration for a few weeks up to over 100
days. Depot preparations can be administered by injection;
parenteral injection; instillation; or implantation, for example,
into soft tissues, a body cavity, or occasionally into a blood
vessel with injection through fine needles.
[0072] Depot formulations can include a variety of bioerodible
polymers including poly(lactide), poly(glycolide),
poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of
desirable lactide:glycolide ratios, average molecular weights,
polydispersities, and terminal group chemistries. Blending
different polymer types in different ratios using various grades
can result in characteristics that borrow from each of the
contributing polymers.
[0073] The use of different solvents (for example, dichloromethane,
chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone,
tetrahydrofuran, phenol, or combinations thereof) can alter
microparticle size and structure in order to modulate release
characteristics.
[0074] Excipients that partition into the external phase boundary
of microparticles such as surfactants including polysorbates,
dioctylsulfosuccinates, poloxamers, PVA, can also alter properties
including particle stability and erosion rates, hydration and
channel structure, interfacial transport, and kinetics in a
favorable manner.
[0075] Additional processing of the disclosed sustained release
depot formulations can utilize stabilizing excipients including
mannitol, sucrose, trehalose, and glycine with other components
such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers
such as Tris, citrate, or histidine. A freeze-dry cycle can also be
used to produce very low moisture powders that reconstitute to
similar size and performance characteristics of the original
suspension.
[0076] Compositions may be formulated for administration locally
via implantation of a membrane, sponge or another appropriate
material onto which the active ingredient has been absorbed or
encapsulated. In certain embodiments, where an implantation device
is used, the device may be implanted into any suitable tissue or
organ, and delivery of the desired active ingredient may be via
diffusion, timed-release bolus, or continuous administration.
Examples include chitosan sponges and collagen sponges.
[0077] In particular embodiments, active ingredients are
administered by a heparin-based delivery system (HBDS), an
affinity-based delivery system that regulates the slow release of
active ingredients by binding them to heparin in fibrin gels. An
HBDS typically contains three main components: (1) a synthetic
linker peptide, (2) the polysulfated glycosaminoglycan heparin, and
(3) the active ingredient(s) to be delivered. Administration of the
compositions may also be achieved by locally supplying active
ingredients using poly(ethylene-co-vinyl acetate) (EVAc)
matrices.
[0078] In particular embodiments, compositions can be formulated
with molecular linkages that facilitate delivery to the central
nervous system (e.g., brain and spinal cord). Particular
embodiments targeting the central nervous system can utilize agents
that bind the transferrin receptor. One example is OX26, a
peptidomimetic MAb that undergoes receptor mediated transcytosis
following binding to the transferrin receptor. See also, e.g., U.S.
Pat. No. 6,372,250B1.
[0079] Nanocarriers can protect therapeutics from degradation
during transport to an active site, and can also aid in transport
across the blood-brain-barrier. Examples of nanocarriers include
liposomes, polymeric nanoparticles, and solid lipid nanoparticles.
Therapeutics can be covalently linked to nanocarriers, or can be
encapsulated without linkages.
[0080] Compositions can be formulated for intranasal delivery.
Intranasal delivery refers to the delivery of a compound to the
nasal passages. When a nasal drug formulation is delivered deep and
high enough into the nasal cavity, the olfactory mucosa can be
reached and drug transport into the central nervous system via the
olfactory receptor neurons can occur. Intranasal administration can
rapidly achieve therapeutic central nervous system concentrations
by delivering therapeutics across the blood-brain-barrier.
Intranasal delivery across the blood-brain-barrier can be achieved
using a propellant device. See for example, US Patent Application
Publication No. 2014/0014104.
[0081] Compositions may be formulated for administration by, or
used in combination with, means of guiding neurite growth, for
example, axonal regrowth to facilitate nerve growth. The neurite
guidance may be used to bridge gaps between nerve endings. One
example is nerve guidance conduits, also referred to as
entubulation, which are composed of biological or synthetic
materials and facilitate communication between proximal and distal
ends of a nerve gap, block external inhibitory factors, and act as
physical axon guidance. In some embodiments, polymer-based active
ingredient delivery may be used with nerve guidance conduits.
Compositions may also be used with longitudinally-oriented
channels, macroscopic structures that can be added to a conduit as
a scaffold. Scaffolds may use materials such as chitosan or
collagen.
[0082] The compositions, kits, and methods may also use
vector-mediated gene delivery techniques to direct expression of
active ingredients. A cell line expressing one or more active
ingredients may be established within the subject or transplanted
to the area of interest, thereby delivering active ingredient to
the affected area. Such a cell line may be a cell line that
endogenously expresses an active ingredient (e.g., IL-17c); may be
a transgenic cell line expressing an active ingredient; or
established by transfection and selection using a vector encoding
an active ingredient. Viral vector-mediated gene delivery may also
be used whereby a viral vector encoding an active ingredient is
delivered directly to the cells of a subject in the area of
interest.
[0083] Any composition disclosed herein can advantageously include
any other pharmaceutically acceptable carriers which include those
that do not produce significantly adverse, allergic, or other
untoward reactions that outweigh the benefit of administration.
Exemplary pharmaceutically acceptable carriers and formulations are
disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack
Printing Company, 1990. Moreover, compositions can be prepared to
meet sterility, pyrogenicity, general safety, and purity standards
as required by U.S. FDA Office of Biological Standards and/or other
relevant foreign regulatory agencies.
[0084] Methods of Use. Methods disclosed herein include treating
subjects (humans, veterinary animals, dogs, cats, reptiles, birds,
etc.), livestock (horses, cattle, goats, pigs, chickens, etc.), and
research animals (monkeys, rats, mice, fish, etc.) with active
ingredients disclosed herein including salts and prodrugs thereof.
Treating subjects includes delivering therapeutically effective
amounts. Therapeutically effective amounts include those that
provide effective amounts, prophylactic treatments, and/or
therapeutic treatments.
[0085] An "effective amount" is the amount of an active ingredient
or composition necessary to result in a desired physiological
change in a subject. Effective amounts are often administered for
research purposes. Effective amounts disclosed herein promote
neural growth and/or neural survival in an animal or clinical
research model of a condition where promotion of neural growth
and/or neural survival is beneficial. For example, exemplary animal
models for diabetic neuropathy include: a streptozotocin
(STZ)-induced diabetes mellitus model in a normal genetic
background in rats; non-obese diabetic (NOD) mice;
Bio-Breeding/Worcester (BB/W) rat; and the Zucker diabetic,
genetically obese rat. Other animal models for diabetes can be
found in Sullivan K A, Lentz S I, Roberts J L, Jr, Feldman E L.
"Criteria for creating and assessing mouse models of diabetic
neuropathy." Current Drug Targets. 2008; 9:3-13. doi:
10.2174/138945008783431763.
[0086] Exemplary animal models for chemical neuropathy use
chemotherapeutic regimens including: taxanes (e.g. docetaxel or
paclitaxel); platinum compounds (e.g. cisplatin, carboplatin, and
oxaliplatin); and others such as vincristine, thalidomide, suramin,
and bortezomib. Chemotherapies can be injected intravenously or
intraperitoneally.
[0087] Exemplary animal models for HIV-associated sensory
neuropathies (HIV-SN) include a transgenic mouse model, where gp120
(the HIV envelope protein) is expressed under the GFAP promoter.
Treatment of the transgenic mouse with didanosine further
accelerates developing neuropathy and results in degeneration of
distal axons of unmyelinated fibers. Injection of toxic
anti-retroviral drugs, such as didanosine or stavudene, into mice
has been used to study neuropathic pain.
[0088] Exemplary animal models for multiple sclerosis include
experimental autoimmune encephalomyelitis (EAE), a family of
disease models, used as the most widely applied means of studying
MS; virus-induced demyelinating disease; toxin induced models of
demyelination (e.g. copper chelator cuprizone); and toxin induced
models of oligodendrocyte death (e.g. diphtheria toxin A).
Exemplary models of spinal cord injury include contusion,
compression and transection models.
[0089] Assays to determine outcome measures in neurodegeneration
animal models include electrophysiological measurements such as
nerve action potentials, and conduction velocity. Skin biopsies,
such as punch biopsies, at various sites are used to evaluate
peripheral neuropathies and can be combined with markers such as
pan-axonal marker, pgp9.5. Intraepidermal nerve fiber density may
also be used, for example, as a true morphological correlate of the
degree of sensory axon loss. Mitochondrial abnormalities may also
be assessed to determine axonal degeneration.
[0090] Behavioral tests that may be used to assess
neurodegeneration include locomotor tests (testing the locomotor
apparatus of the animal); motor tests (analyzing the strength,
coordination and other abilities of the skeletal muscles); sensory
tests (evaluating proprioception, touch, pain or temperature
sensing); sensory-motor tests; (testing the proper connection
between the sensory and motor systems); autonomic tests;
(evaluating the function of the sympathetic and parasympathetic
systems); and reflex response based tests. Sensory behavioral
testing includes evaluating sensations such as thermal hyperalgesia
by the tail flick test and the hot plate method; mechanical
hyperalgesia; mechanical allodynia; and chemical allodynia.
[0091] A "prophylactic treatment" includes a treatment administered
to a subject who does not display signs or symptoms of
neurodegeneration; or only displays early signs or symptoms of
neurodegeneration such that treatment is administered for the
purpose of promoting neural growth and/or neural survival for the
purpose of diminishing, preventing, or decreasing the risk of
developing neurodegeneration further. Thus, a prophylactic
treatment functions as a preventative treatment against
neurodegeneration.
[0092] A "therapeutic treatment" includes a treatment administered
to a subject who displays symptoms or signs of neurodegeneration,
and is administered to the subject for the purpose of promoting
neural growth and/or neural survival to alleviate symptoms
associated with the neurodegeneration.
[0093] Symptoms of neurodegeneration can include disruptions or
conditions of the somatosensory system, including disruptions of
nociception, mechanoreception, proprioception, and thermoreception.
Symptoms of neurodegeneration can also include hyperesthesia (an
abnormal increase in sensitivity to a sensory stimulus, such as the
sensation of pain in response to a stimulus that is normally not
painful), hypoesthesia (reduced sensation, or a partial loss of
sensitivity to sensory stimuli), anesthesia (a lack of sensation)
paresthesia (abnormal sensation, including the sensations of
tingling, tickling, pricking, burning, or stabbing pain without
corresponding sensory stimulus), hyperalgesia (increased
sensitivity to pain), and allodynia (sensation of pain due to a
non-noxious stimulus). These sensory disturbances may be
characterized further by their location, pattern of onset,
consistency, and factors that exacerbate or alleviate symptoms.
[0094] Symptoms of neurodegeneration also can include neuropathic
pain, often the result of peripheral neuropathies. Neuropathic pain
refers to pain that originates from pathology of the nervous
system. Neuropathic pain may result from lesions of the nervous
system. Abnormal signals arise not only from injured axons but also
from the intact nociceptors that share the innervation territory of
an injured nerve. The nervous system can generate and perpetuate
pain without any ongoing stimuli from injury. Neuropathic pain is
often puzzling and frustrating for both patients and physicians
because it seems to have no cause; responds poorly to standard pain
therapies; can last indefinitely and even escalate over time; and
often results in severe disability.
[0095] Additional symptoms of neurodegeneration can be paralysis,
difficulty of movement, speech impairment, tremors, and cognitive
impairment. Causes of neurodegeneration in the central nervous
system include spinal cord injury and multiple sclerosis, for
example.
[0096] Additional exemplary degenerative nerve diseases or
conditions leading to neurodegeneration include Alzheimer's
disease; amyotrophic lateral sclerosis; Friedreich's ataxia;
Huntington's disease; Lewy body disease; Parkinson's disease; and
spinal muscular atrophy. Exemplary motor neuron diseases or
conditions leading to neurodegeneration include amyotrophic lateral
sclerosis (ALS), also called Lou Gehrig's disease; progressive
bulbar palsy; pseudobulbar palsy; primary lateral sclerosis (PLS);
progressive muscular atrophy; spinal muscular atrophy (SMA); and
post-polio syndrome (PPS).
[0097] Neurodegeneration can be idiopathic. Neurodegeneration can
also be small fiber neuropathies. Neurodegeneration can be caused
by alcoholism; bone marrow disorders (e.g., abnormal monoclonal
gammopathies, amyloidosis, osteosclerotic myeloma, lymphoma);
diseases (e.g., autoimmune diseases (e.g., chronic inflammatory
demyelinating polyneuropathy, Guillain-Barre syndrome, lupus,
multiple sclerosis, spinal cord injury, necrotizing vasculitis,
rheumatoid arthritis, Sjogren's syndrome); connective tissue
disorders; diabetes mellitus; hypothyroidism; kidney disease; liver
disease; exposure to poisons or toxins (e.g., heavy metals or
chemicals); infections (e.g., viral or bacterial including Lyme
disease, shingles (i.e. varicella-zoster), Epstein-Barr virus,
hepatitis C, herpes, leprosy, diphtheria and human immunodeficiency
virus (HIV)); injuries (e.g., from nerve pressure (from, e.g.,
cancerous and noncancerous growths on the nerves themselves, or in
an area that puts pressure on surrounding nerves), repetitive
motion or trauma); inherited causes or disorders (e.g.,
Charcot-Marie-Tooth disease); medical conditions; medical
treatments (e.g., chemotherapy or radiation therapy); medications;
metabolic problems; and vitamin deficiencies (e.g., vitamin B-1,
B-3, B-6, B-12, E).
[0098] Therapeutically effective amounts generating promotion of
neural growth and/or neural survival can be evidenced by increased
neurite growth (e.g., axon and/or dendrite growth), neurite
guidance in a particular direction, increased branching points of
neurites or nerve fibers, increased innervation, neural cell
survival, neural cell regeneration, nerve cell or nerve fiber
density, nerve fiber growth, nerve fiber length, or decreased
apoptosis, degeneration of cells, neurite degeneration, or neural
cell degeneration.
[0099] Subjects also may be assessed for promotion of neural growth
and/or neural survival by a number of accepted procedures known in
the art including electrodiagnostic tests such as Nerve Conduction
Studies and Electromyography (EMG); and Nerve Conduction Velocity
tests that evaluate how nerves transmit electrical stimuli by
measuring the speed of conduction of an electrical impulse. These
tests can help determine whether neurodegeneration involves axons
or myelin. EMG measures the electrical activity of muscles in
response to nerve stimulations. Skin Biopsy may also be used to
measure nerve fibers in the skin and to identify specific
neuropathies, such as small fiber neuropathy. In particular, skin
punch biopsy may be used at standard sites to measure the density
of the small nerve fibers, as determined by morphometry after
immunostaining, for example, with an antibody to the axonal marker
pgp 9.5.
[0100] Autonomic Tests also may be used to assess neural growth
and/or neural survival, including the Quantitative Sudomotor Axon
Reflex Test (QSART) and the tilt table test. QSART measures the
autonomic nerve fibers that stimulate sweating. The tilt table test
measures changes in blood pressure and pulse from prone to vertical
positions.
[0101] Tests used to assess neural growth and/or neural survival
related to neurodegeneration include symptoms, signs or evidence
indicating disease of the brain or spinal cord. Evidence of two or
more lesions, or abnormal areas in the brain, using a Magnetic
Resonance Imaging (MRI) scan may be identified. Evoked potential
tests may be performed to measure the time it takes to respond to
stimulation (i.e. visual, auditory and somatosensory). Tests for
infection, for example, in cerebrospinal fluid may also be
administered. Spinal cord injury may be identified by computerized
tomography (CT) scan; x-ray; and MRI.
[0102] Therapeutically effective amounts generating promotion of
neural growth and/or neural survival also can be evidenced by
reduction in a symptom associated with neurodegeneration.
[0103] In various embodiments, the compositions, kits, and methods
are used to promote neural growth and/or neural survival in the
autonomic nervous system, the central nervous system, the
parasympathetic nervous system, the peripheral nervous system, the
sensory nervous system, and/or the sympathetic nervous system.
[0104] In various embodiments, the compositions, kits, and methods
are used to promote neural growth and/or neural survival of neural
progenitor cells, autonomic nerve fibers, motor nerve fibers, motor
neurons, proprioceptive sensory fibers, sensory nerve fibers,
and/or sensory neurons.
[0105] In particular embodiments, the compositions, kits, and
methods may be used in the dermis, epidermis or at the
dermal-epidermal junction.
[0106] Administering in or around a site of means within 5 inches
of a site of interest; within 4 inches of a site of interest;
within 3 inches of a site of interest; within 2 inches of a site of
interest; or within 1 inch of a site of interest.
[0107] In various embodiments, the compositions, kits, and methods
may be used in combination with other treatments including
treatment of the underlying cause of the neurodegeneration, such as
a vitamin deficiency, an infection, a neurodegenerative disorder
(e.g., Alzheimer's disease, Parkinson's disease), or a procedure or
treatment such as surgery or chemotherapy. In particular
embodiments, they may be used in combination with surgical removal
of a tumor that put pressure on surrounding nerves.
[0108] A subject in need of promotion of neural growth and/or
neural survival can be a subject that has been assessed for
neurodegeneration and found to have a symptom of the
neurodegeneration or has been determined to be at risk for
developing neurodegeneration. Methods for assessing subjects for
neurodegeneration include any art-accepted test including the
exemplary art-accepted tests disclosed herein. For example, a
subject may be assessed for symptoms including sensory disturbances
by positive features (too much sensation, spontaneous sensation,
etc.), or negative sensory deficits (too little sensation or
numbness). A subject in need of promotion of neural growth and/or
neural survival can also be a research animal undergoing
experimental procedures in an animal model of neurodegeneration
(e.g., spinal cord injury or diabetic neuropathy).
[0109] Assessment of a subject for neurodegeneration can include
evaluation of a subject's medical history using information that
may include past medical conditions; family history; symptom onset;
progression and pattern of involvement; co-existing medical
conditions; previous treatments; and medications. Assessment may
also involve a neurological examination to evaluate motor and
sensory nerve functions; strength and sensation; balance;
coordination; and reflexes, as well as evaluation of biomarkers of
nerve presence and function (e.g., MRI, PET, and radionucleotide
imaging).
[0110] The actual dose amount administered to a particular subject
can be determined by a physician, veterinarian, or researcher
taking into account parameters such as physical and physiological
factors including target area; body weight; severity of
neurodegeneration or resulting condition; prospective conditions;
type of neural cells or neurites requiring growth and/or survival
promotion; previous or concurrent therapeutic interventions;
idiopathy of the subject; and route of administration.
[0111] The amount and concentration of an active ingredient(s) in a
composition, as well as the quantity of the composition
administered to a subject, can be selected based on clinically
relevant factors, the solubility of the active ingredient in the
composition, the potency and activity of the active ingredient, and
the manner of administration of the composition, as well as whether
the active ingredient is modified (e.g., nitrited, PEGylated) or
administered in combination with other treatments.
[0112] A composition including a therapeutically effective amount
of an active ingredient(s) disclosed herein can be administered to
a subject for promoting neural growth and/or neural survival in a
clinically safe and effective manner, including one or more
separate administrations of the composition.
[0113] Useful doses can often range from 0.1 to 5 .mu.g/kg. Other
doses can range from 1-2 mg/kg, 1-5 mg/kg, 1-10 mg/kg, 1-15 mg/kg,
1-25 mg/kg, 1-50 mg/kg, 1-55 mg/kg, 1-100 mg/kg, 1-250 mg/kg, 1-500
mg/kg, 1-750 mg/kg, or 1-1000 mg/kg. In other examples, a dose can
include 1 .mu.g/kg, 10 .mu.g/kg, 50 .mu.g/kg, 75 .mu.g/kg, 100
.mu.g/kg, 150 .mu.g/kg, 200 .mu.g/kg, 500 .mu.g/kg, 1000 .mu.g/kg,
0.1 to 5 mg/kg, or from 0.5 to 1 mg/kg. In other examples, a dose
can include 1 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg, 55 mg/kg, 100
mg/kg, 250 mg/kg, 500 mg/kg, 750 mg/kg, 1000 mg/kg, or more.
[0114] Each of the described doses of active ingredients can be an
active ingredient alone, or in combination of one or more other
active ingredients. In particular embodiments, when included in
combinations to produce a dose, such as a dose stated herein, the
substituents in the combination can be provided in exemplary ratios
such as: 1:1; 1:1.25; 1:1.5; 1:1.75; 1:8; 1:1.2; 1:1.25; 1:1.3;
1:1.35; 1:1.4; 1:1.5; 1:1.75; 1:2; 1:3; 1:4; 1:5; 1:6:1:7; 1:8;
1:9; 1:10; 1:15; 1:20; 1:30; 1:40; 1:50; 1:60; 1:70; 1:80; 1:90;
1:100; 1:200; 1:300; 1:400; 1:500; 1:600; 1:700; 1:800; 1:900;
1:1000; 1:1:1; 1:2:1; 1:3:1; 1:4:1; 1:5;1; 1:10:1; 1:2:2; 1:2:3;
1:3:4; 1:4:2; 1:5;3; 1:10:20; 1:2:1:2; 1:4:1:3; 1:100:1:1000;
1:25:30:10; 1:4:16:3; 1:1000:5:15; 1:2:3:10; 1:5:15:45;
1:50:90:135; 1:1.5:1.8:2.3; 1:10:100:1000 or additional beneficial
ratios depending on the number and identity of substituents in a
combination to reach the stated dosage. The substituents in a
combination can be provided within the same composition or within
different compositions.
[0115] Therapeutically effective amounts can be achieved by
administering single or multiple doses during the course of a
treatment regimen (e.g., QID, TID, BID, daily, every other day,
every 3 days, every 4 days, every 5 days, every 6 days, weekly,
every 2 weeks, every 3 weeks, monthly, every 2 months, every 3
months, every 4 months, every 5 months, every 6 months, every 7
months, every 8 months, every 9 months, every 10 months, every 11
months, or yearly).
[0116] Compositions may be administered before an upcoming insult,
such as administration of chemotherapy, radiation, or medications
that may cause neurodegeneration. In particular embodiments,
compositions are administered within 10 days of an upcoming insult,
within 9 days of an upcoming insult, within 8 days of an upcoming
insult, within 7 days of an upcoming insult, within 6 days of an
upcoming insult, within 5 days of an upcoming insult, within 4 days
of an upcoming insult, within 3 days of an upcoming insult, within
48 hours of an upcoming insult, within 46 hours of an upcoming
insult, within 44 hours of an upcoming insult, within 42 hours of
an upcoming insult, within 40 hours of an upcoming insult, within
38 hours of an upcoming insult, within 36 hours of an upcoming
insult, within 34 hours of an upcoming insult, within 32 hours of
an upcoming insult, within 30 hours of an upcoming insult, within
28 hours of an upcoming insult, within 26 hours of an upcoming
insult, within 24 hours of an upcoming insult, within 22 hours of
an upcoming insult, within 20 hours of an upcoming insult, within
18 hours of an upcoming insult, within 16 hours of an upcoming
insult, within 14 hours of an upcoming insult, within 12 hours of
an upcoming insult, within 10 hours of an upcoming insult, within 8
hours of an upcoming insult, within 6 hours of an upcoming insult,
within 4 hours of an upcoming insult, or within 2 hours of an
upcoming insult. In one particular embodiment, compositions are
administered within 18 hours of an upcoming insult.
[0117] Also disclosed herein are kits including one or more
containers including one or more of the active ingredients and/or
compositions described herein. In various embodiments, the kits may
include one or more containers containing one or more active
ingredients and/or compositions to be used in combination with the
active ingredients and/or compositions described herein. Associated
with such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use, or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use, or sale for human
administration.
[0118] Optionally, the kits described herein further include
instructions for using the kit in the methods disclosed herein. In
various embodiments, the kit may include instructions regarding
preparation of the active ingredients and/or compositions for
administration; administration of the active ingredients and/or
compositions; appropriate reference levels to interpret results
associated with using the kit; proper disposal of the related
waste; and the like. The instructions can be in the form of printed
instructions provided within the kit or the instructions can be
printed on a portion of the kit itself. Instructions may be in the
form of a sheet, pamphlet, brochure, CD-Rom, or computer-readable
device, or can provide directions to instructions at a remote
location, such as a website. The instructions may be in English
and/or in any national or regional language. In various
embodiments, possible side effects and contraindications to further
use of components of the kit based on a subject's symptoms can be
included. The kits and instructions can also be tailored according
to the area of the body to be treated.
[0119] In various embodiments, the packaging, active ingredients
and/or compositions, and instructions are combined into a small,
compact kit with printed instructions for use of each of the active
ingredients and/or compositions. In various embodiments in which
more than one active ingredient and/or composition is provided, the
sequencing of use of the active ingredients and/or compositions can
be labeled in the kit.
[0120] In various embodiments, the kits described herein include
some or all of the necessary medical supplies needed to use the kit
effectively, thereby eliminating the need to locate and gather such
medical supplies. Such medical supplies can include syringes,
ampules, tubing, facemask, a needleless fluid transfer device, an
injection cap, sponges, sterile adhesive strips, Chloraprep,
gloves, and the like. Variations in contents of any of the kits
described herein can be made.
[0121] The Exemplary Embodiments and Examples below are included to
demonstrate particular embodiments of the disclosure. Those of
ordinary skill in the art should recognize in light of the present
disclosure that many changes can be made to the specific
embodiments disclosed herein and still obtain a like or similar
result without departing from the spirit and scope of the
disclosure.
Exemplary Embodiments
[0122] 1. A method of promoting neural growth and/or neural
survival in a subject including administering to the subject a
therapeutically effective amount of an IL-17c protein thereby
promoting neural growth and/or neural survival in the subject 2. A
method of embodiment 1, wherein the neural growth is evidenced by
nerve density, neurite growth (axon or dendrite) and/or neurite
(axon or dendrite) guidance. 3. A method of embodiments 1 or 2,
wherein the IL-17c protein includes SEQ ID NO: 1. 4. A method of
any of embodiments 1-3, wherein the promoted neural growth and/or
neural survival is found in a sensory or motor neural cell and/or
nerve. 5. A method of any of embodiments 1-4, wherein the
administering is in or around a site of the sensory or motor neural
cell and/or nerve. 6. A method of any of embodiments 1-5, wherein
the administering is topical. 7. A method of any of embodiments
1-6, wherein the administering is through application of a
transdermal patch. 8. A method of any of embodiments 1-7, wherein
the administering is prophylactic. 9. A method of any of
embodiments 1-8, wherein the administering is before an upcoming
insult. 10. A method of embodiment 9, wherein the upcoming insult
is a scheduled insult. 11. A method of embodiment 10, wherein the
scheduled insult is surgery or chemotherapy. 12. A method of any of
embodiments 1-11 wherein the promoting alleviates a symptom of
neurodegeneration. 13. A method of embodiment 12 wherein the
neurodegeneration is a peripheral neuropathy. 14. A method of
promoting neural growth and/or neural survival including contacting
a neural cell or nerve with a therapeutically effective amount of
an IL-17c protein thereby promoting neural growth and/or neural
survival. 15. A method of embodiment 14, wherein the neural growth
and/or neural survival is evidenced by increased neural cell
survival, increased neurite growth (axon or dendrite), neurite
guidance (axon or dendrite), and/or increased innervation 16. A
method of embodiment 14 or 15, wherein the IL-17c protein includes
SEQ ID NO: 1. 17. A method of any of embodiments 14-16, wherein the
neural cell or nerve is from the peripheral nervous system. 18. A
method of any of embodiments 14-17, wherein the neural cell or
nerve is from a sensory or motor neural cell or nerve. 19. A method
of any of embodiments 14-18, wherein the neural cell or nerve is
within the dermis of a subject. 20. A method of embodiment 19,
wherein the subject is a subject in need of the promoting neural
growth and/or neural survival. 21. A method of any of embodiments
14-20, wherein the promoting alleviates a symptom of
neurodegeneration. 22. A method of any of embodiments 14-20,
wherein the neurodegeneration is a peripheral neuropathy. 23. A
composition including a therapeutically effective amount of an
IL-17c protein and a pharmaceutically acceptable carrier. 24. A
composition of embodiment 23, wherein the IL-17c protein includes
SEQ ID NO: 1. 25. A composition of embodiment 23 or 24, wherein the
pharmaceutically acceptable carrier includes a topical carrier. 26.
A composition of any of embodiments 23-25, wherein the
pharmaceutically acceptable carrier is selected from an aqueous
carrier, an oil-based carrier, a fat-based carrier, a fatty
alcohol-based carrier, or a combination thereof. 27. A kit for
promoting neural growth and/or neural survival, the kit including a
therapeutically effective amount of an IL-17c protein and
instructions for administering the therapeutically effective amount
of the IL-17c protein to a subject. 28. A method of promoting
neural growth and/or neural survival including contacting
keratinocytes with a therapeutically effective amount of a virus, a
TLR2 ligand and/or a TLR5 ligand thereby promoting neural growth
and/or neural survival. 29. A method of promoting neural growth
and/or neural survival including contacting keratinocytes with a
virus, a TLR2 ligand and/or a TLR5 ligand in an amount sufficient
to elicit release of a therapeutically effective amount of IL-17c
thereby promoting neural growth and/or neural survival. 30. A
method of embodiment 28 or 29 wherein the virus is HSV, the TLR2
ligand is peptidoglycan or the TLR5 ligand is flagellin. 31. A
method of embodiments 28-30, wherein the neural growth and/or
neural survival is evidenced by increased neural cell survival,
increased neurite growth (axon or dendrite), neurite guidance (axon
or dendrite), and/or increased innervation within 5 inches of the
contacted keratinocytes. 32. A method of embodiments 28-31, wherein
the neural cell or nerve is from the peripheral nervous system. 33.
A method of embodiments 28-32, wherein the neural cell or nerve is
from a sensory or motor neural cell or nerve. 34. A method of
embodiments 28-33, wherein the neural cell or nerve is within the
dermis of a subject. 35. A method of embodiment 34, wherein the
subject is a subject in need of the promoting neural growth and/or
neural survival. 36. A method of any of embodiments 28-35, wherein
the promoting alleviates a symptom of neurodegeneration. 37. A
method of any of embodiments 28-36, wherein the neurodegeneration
is a peripheral neuropathy. As stated, IL-17c proteins include SEQ
ID NO: 1 and biologically active analogues thereof (e.g., variants,
D-substituted analogs and modifications). Reference to other active
ingredients also includes biologically active analogues of the
referenced active ingredient.
Example 1
[0123] Introduction. Keratinocytes, immune cells and nerve fibers
are interconnected anatomically and functionally in skin (Chuong et
al., 2002; Misery, 1997). Herpes simplex virus types 1 and 2 (HSV-1
& HSV-2) have evolved strategies to exploit this system for
recurrent infection. After primary infection at the site of
acquisition (mouth and genitals), viruses travel retrogradely via
axons to cell bodies of peripheral sensory neurons where they
establish latency. Reactivation from latency involves anterograde
movement to sites near the original site of entry for replication
and transmission (Roizman and Whitley, 2013). Human recurrent HSV-2
infection is frequent and often clinically asymptomatic (Johnston
et al., 2012; Schiffer et al., 2013; Wald et al., 1997). While
sensory anesthesia may precede or accompany HSV-2 reactivation,
reports of such peripheral nerve damage or neuropathy are extremely
rare among patients with HSV-2 recurrent infection, a clinical
observation that distinguishes it markedly from Varicella Zoster
Virus infection, where nerve destruction and neuropathy are well
recognized. It is unclear how peripheral nerves maintain their
function in spite of frequent HSV-2 reactivation over time. In
fact, there is controversy whether peripheral nerve damage is
associated with human HSV-2 reactivation.
[0124] The interleukin 17 (IL-17) family consists of 6 members
(IL-17a, IL-17b, IL-17c, IL-17d, IL-17e and IL-17f) (Gaffen, 2009;
Gaffen, 2011). To date IL-17c has been identified as an epithelial
cell derived cytokine that regulates innate immune function
(Ramirez-Carrozzi et al., 2011; Song et al., 2011) and promotes
inflammation in psoriasis (Johnston et al., 2013; Ramirez-Carrozzi
et al., 2011). Here it is reported that both keratinocytes and
neurons produce IL-17c in response to HSV-2 infection and IL-17c
functions as a neurotrophic factor that can provide a survival
signal to protect neurons from apoptosis during HSV infection and
most importantly stimulates peripheral nerve growth.
[0125] Methods. Study participants. Healthy, HSV-2 seropositive
adults were recruited at the University of Washington Virology
Research Clinic in Seattle, Wash. HSV-2 serostatus was determined
by Western blot as previously described (Koutsky et al., 1992); all
participants were HIV seronegative and biopsy procedures were
conducted as described previously (Peng et al., 2009; Zhu et al.,
2007). The biopsy protocol was approved by University of Washington
Human Subjects Review Committee and all participants provided
written consent. All samples were immediately placed on dry ice and
stored at -80.degree. C. until processing.
[0126] Purification of keratinocytes from genital skin biopsies. A
rapid immunofluorescent staining method (<15 minutes) was
utilized to identify CD8+ T cells located at the dermal epidermal
junction (DEJ) from skin biopsies (Peng et al., 2012; Zhu et al.,
2013). Then the Zeiss PALM Microbeam laser capture micro-dissection
(LCM) system was used to cut and catapult individual keratinocytes
above the basement membrane to designated tubes in a completely
automated process. Between 50 and 100 cells were captured per skin
biopsy and 1 to 10 ng of isolated total RNA was processed for gene
expression analysis via the Illumina array platform.
[0127] RNA extraction, amplification and hybridization of cDNA to
Illumina beadarrays. Total RNA from LCM-captured keratinocytes was
extracted using PicoPure RNA isolation kits following the
manufacturer's protocol (Applied Biosystems, CA, USA). The quality
of total RNA was analyzed by Agilent pico chips and RNA with a
quality index (RIN) above 5 was used. Total RNA (0.5-1 ng) was then
used for cDNA synthesis using the Ovation Pico RNA Amplification
System (NuGEN, CA, USA). The size distribution of cDNA was analyzed
by Agilent Technologies nano chips and the amplified cDNA had a
Gaussian distribution with an average size of 200 bp. The cDNA was
biotin-labeled per the NuGEN protocol and labeled cDNA (750 ng) was
hybridized to Illumina HT-12 beadarrays in the Shared Resource
Genome Center at Fred Hutchison Cancer Research Center per the
manufacturer's instructions.
[0128] Analysis of beadarray data. Raw data were imported to
GenomeStudio (V2010.3, Illumina). Control summaries were generated
to analyze the quality of hybridization. Data passing this initial
quality control step were normalized using Cubic Spline with
background subtraction. Normalized data were exported to R and
differentially expressed genes between keratinocytes from control
biopsies and those from healed skin biopsies were selected using
Genefilter, a Bioconductor package. The differentially expressed
genes were analyzed using an unsupervised hierarchical clustering
method (Clustering method: UPGMA [weighted average] and similarity
measure: euclidean distance) using SpotFire DecisionSite for
functional genomics (Version 9.1.2). Enriched functional categories
and network analyses for differentially expressed genes were
performed using Ingenuity Pathway Analysis (IPA 8.8). The GOMiner
program was used to annotate all the 20,818 genes on Illumina Human
HT-12 beadarrays. An annotation database was constructed in
Microsoft Access using exported tables from GOMiner and genes that
were annotated to the following GO terms: cytokine/chemokine/growth
factor activity and cytokine/chemokine/growth factor receptor
activity were exported for further analysis in SpotFire.
[0129] Viral stocks. Viral stocks utilized in this study include
HG52 (HSV-2); HSV-1 strains are KOS and ICP8 mutant (ICP8mu)
(kindly provided by Dr. David Knipe, Harvard Medical School,
Boston, Mass.), ICP0 mutant (ICP0mu) and ICP22 mutant (ICP22mu)
(kindly provided by Dr. William P. Halford, Southern Illinois
University School of Medicine, Springfield, Ill.) and K26 which
contains VP26-GFP fusion gene (a generous gift from Dr. Prashant
Desai, Johns Hopkins University, Baltimore, Md.). Viral titers were
determined by titration in Vero cells.
[0130] Cell cultures. Primary keratinocytes were purchased from
Lifeline Cell Technology (Frederick, Md.). Cells were cultured in
DermaLife.RTM. Basal Medium with DermaLife K LifeFactors (Cat #
LS-1030) as recommended by the manufacturer. Acyclovir stock
solution was prepared in DMSO at 6.76 mg/ml (30 mM) (Sigma) and
diluted 1:1000 for use on primary keratinocytes (30 .mu.M). To UV
treat a virus stock, HG52 virus stock was spread on a tissue
culture grade 60 mm petri dish.
[0131] With the petri dish lid off a UV light source was placed 2
inches above virus for 30 minutes. UV-treated virus was stored at
-80.degree. C. for later use. For TLR2 and TLR5 stimulation, cells
were treated with peptidoglycan (PGN) at 2 .mu.g/ml or flagellin at
0.1 .mu.g/ml (PGN and flagellin are from InvivoGen and Sigma,
respectively). The TLR2 neutralizing antibody is from InvivoGen. To
block IL-17c signaling, keratinocytes were treated with a
neutralizing antibody for human IL-17RA (mouse monoclonal antibody)
and matching mouse control IgG (R&D systems) (2 .mu.g/ml) for 1
hour before HSV infection.
[0132] Primary MCN were purchased from Life Technologies and
cultured as recommended by the manufacturer. To block IL-17c
signaling, cells were treated with a neutralizing antibody for
IL-17RA (rat monoclonal antibody) and matching rat control IgG
(R&D systems) (2 .mu.g/ml) for 1 hour before K26 infection.
Murine IL-17c (mIL17c) was synthesized and purified in Fred
Hutchinson Research Center shared resource facility. To detect
apoptosis during HSV infection of mouse primary neurons, cells were
stained with an antibody for cleaved caspase 3 (Cell Signaling
Technology) according to the manufacturer's methods.
[0133] Human SH-SY5Y neuroblastoma cells were obtained from the
ATCC. The SH-SY5Y neuroblastoma cell lines were maintained in 1:1
mixture of ATCC-formulated EMEM and F12 media containing 15% (v/v)
heat-inactivated FBS without antibiotics. SH-SY5Y cells were
induced to differentiate up to 7 days with 50 .mu.m all-trans
retinoic acid (ATRA; Sigma) in 1:1 mixture EMEM/F12 media
supplemented with 5% (v/v) FBS without antibiotics. They were
monitored daily by phase-contrast microscopy for the appearance of
elongated neurites. A differentiated cell was defined as a cell
with a neurite length greater than the length of the cell body (on
average greater than 10 .mu.m in length) and expressing
.beta.-tubulin III (Abcam).
[0134] Isolation of Human Fetal Dorsal Root Ganglia and Sensory
Neurons. Human fetal spinal cords were isolated from first and
early second trimester aborted specimens, obtained from the
Laboratory of Developmental Biology in full compliance with the
ethical guidelines of the National Institutes of Health and with
the approval of the University of Washington institutional review
boards for the collection and distribution of human tissues for
research. The Laboratory of Developmental Biology obtained written
consent from all tissue donors. The tissue was briefly washed in
Hanks' balanced salt solution (HBSS) and transported in Hibernate E
at 4.degree. C. prior to isolation of DRG.
[0135] All ganglia were dissected under sterile conditions,
dissected free of fascia and connective tissue, and collected in
DMEM and digested in 0.25% trypsin solution for 30 min at
37.degree. C., washed in culture medium containing 10% FBS, and
then triturated into a single-cell suspension with a fire-polished
glass Pasteur pipet. Cells were resuspended in Neurobasal media
(NB, Life Technologies) supplemented with B27 and 0.5 mM Glutamax
(with antibiotics) and counted with trypan blue assay. Cell
suspensions were plated in Poly-D-Lysine and Laminin coated 8-well
chamber slides (BD/Corning) in NB/B27 media supplemented with 50
ng/ml human .beta.-NGF (Millipore) and incubated overnight at
37.degree. C. For each experiment, the cells were washed twice with
basal media before addition of PBS or recombinant human IL-17c (200
ng/mL, ebioscience; or internally-generated human IL-17c). Cells
with neurites were scored as neurons. Identification of the cells
as neurons was confirmed by showing reactivity on the neurites with
monoclonal antibody for PGP9.5.
[0136] Culture of neurons in microfluidic chambers. Microfluidic
chambers (Xona Microfluidic, LLC) were autoclaved and bonded into
FluoroDish (World Precision Instruments, Inc.) using a laboratory
Corona treater (Electro-Technic Products, Inc.). Microfluidic
chambers were coated with 1% () polyethylenimine (PEI) for 10 mins
and 0.1% () glutaraldehyde for 30 mins to provide adhesion to the
collagen gels. Rat tail collagen I (Gibco) was perfused through
microfluidic chambers at a concentration of 5 .mu.g/cm.sup.2.
Twenty microliters of 2,000,000 cells/mL differentiated SY5Y cells
were seeded into the soma channel.
[0137] After 10 mins, 50 .mu.l of culture media was added into each
soma reservoir and 70 .mu.l of culture media with 200 ng/ml IL-17c
or NGF or same volume of PBS was added into each distal reservoir.
Half of the growth media was changed every other day. Ten days
later, cultures were fixed and stained with a PGP9.5 antibody.
[0138] For human primary fetal neuron culture, 3-channel
microfluidic chambers (Xona Microfluidic, LLC) were autoclaved and
bonded onto clean coverslips (Corning) using a plasma cleaner
(Harrick Scientific, Inc.). The chambers were sterilized with 70%
ethanol and then coated with 0.5 mg/mL poly-d-lysine (Corning) and
10 .mu.g/ml mouse laminin (Life Technologies). Dissociated human
fetal DRG neurons were plated into the middle channel at a density
of 100,000 cells/chamber. After 4 days, 200 ng/mL IL-17c was added
into the right channel to generate a gradient of IL-17c in the soma
(middle) channel. Half of the growth media was changed every other
day. Sixteen days later, cultures were fixed and stained.
[0139] Time-lapse microscopy. Transmitted light time lapse
microscopy of neuroblastoma cells in microfluidic devices was
performed on a Nikon Ti inverted microscope fitted with a Nikon
40.times./0.9 S Fluor objective (Nikon Instruments Inc., Melville,
N.Y.) and a Photometrics Coolsnap HQ scientific grade CCD camera.
The devices were mounted inside a Chamlide stage top incubator
(Live Cell Instrument, Seoul, Korea) maintained at 37.degree. C.
and 5% CO.sub.2 and focus was maintained with Nikon's proprietary
Perfect Focus system. Bright field images were collected at 5 min
intervals for 16 hours.
[0140] Long term kinetic imaging of cultivated human neurons grown
in 8-well chamber slides inside a conventional tissue culture
incubator was performed with an Incucyte microscope system (Essen
Bioscience Inc., Ann Harbor, Mich.) fitted with a Nikon
10.times./0.3 Plan Fluor objective. Bright field images were
collected at one-hour intervals. Four fields of view were imaged in
phase contrast for each well, and average neurite length and number
of branch points were measured with the Incucyte neuro-track image
analysis software module.
[0141] Slide scanning and cytometric analysis. To quantify IL-17c+
cells, 30,000 keratinocytes or 80,000 MCN were cultured overnight
or for 3 days in 8 well chamber slides before IL-17c pre-treatment
and K26 infection (MOI of 2 and 5 for keratinocytes and MCN,
respectively). Human keratinocytes were treated with human IL-17c
(hIL-17c) (eBioscience) at 200 ng/mL in the presence of human
IL-17RA neutralizing antibody (2 .mu.g/mL) or matching control
mouse IgG (2 .mu.g/mL) for 12 hours before K26 infection. MCN were
pretreated with murine IL-17c (mIL-17c) at 20 ng/mL in the presence
of murine IL-17RA neutralizing antibody (2 .mu.g/mL) or matching
control rat IgG (2 .mu.g/mL) for 24 hours before infection. Slides
were scanned on a Tissuefax microscope system (Tissuegnostics GmbH,
Vienna, Austria) including a Zeiss Imager Z2 upright fluorescence
microscope, motorized Marzhauser stage with 8-slide capacity, and
PCO pixelfly QE CCD camera. System operation was controlled by
Tissuefax software which provided automated large area acquisition
with image stitching and autofocus. Images were acquired with a
Zeiss Plan Apochromat 10.times./0.45 objective. Zeiss fluorescence
filter sets for DAPI, FITC (for GFP) and Cy5 (for IL-17c and
cleaved caspase 3) were used.
[0142] Image analysis was performed with Tissuegnostics TissueQuest
software. Whole slide scans were imported into TissueQuest. Images
were segmented and cells were identified by setting appropriate
intensity thresholds and cell size parameters for all channels.
Live cells were identified and counted based on nuclear channel
staining (DAPI). Average staining intensity of the green and far
red channels was measured for all segmented objects (cells).
Typically, a cell mask including nucleus and cytoplasm was used. In
some cases where the staining was predominantly cytoplasmic and
cell size and shape was very heterogeneous, a ring mask derived
from the nuclear mask was used to sample average staining intensity
in the cytoplasm. Once all cell data had been obtained, intensity
values for the desired channels were plotted for all cells as
density plot using sm package in R and appropriate cut-offs were
set to obtain counts and percentages of positive cells. DAPI+ cells
were counted as live neurons and cleaved caspase 3+ cells were
counted as neurons under apoptosis. The accuracy of the algorithms
was verified by performing manual counts of selected regions and
comparing them with the output of the TissueQuest software; there
was good agreement between the two methods.
[0143] Immunofluorescent staining. The staining methods were
previously described (Peng et al., 2012; Zhu et al., 2009; Zhu et
al., 2007; Zhu et al., 2013). The antibodies for staining were
purchased from the following sources: IL-17c antibody (mouse
monoclonal, R&D); IL-17RE antibody (rabbit polyclonal, Sigma);
IL-17RA antibody (rabbit monoclonal, LifeSpanBioSciences); NCAM
antibody (mouse monoclonal, Bechman Coulter); PGP9.5 (Abcam); NF200
antibody (rabbit polyclonal, Sigma); Peripherin antibody (rabbit
& mouse, Sigma); NF-.kappa.B and IRF-3 antibodies (rabbit
polyclonal and mouse monoclonal, respectively, Santa Cruz); Cleaved
caspase 3 antibody (rabbit polyclonal, Cell Signaling).
[0144] Quantitative RT-PCR (qRT-PCR) assay. Total RNA was extracted
from human primary keratinocytes and mouse cortical neurons using
Qiagen RNeasy mini kits. The cDNA was synthesized from total RNA
using high capacity cDNA synthesis kits (Applied Biosystem). The
TaqMan probes for ACTB, IL-17c, IL-17RE, NF-.kappa.B, IRF1, IRF3,
IRF7, IFI16 and PML were ordered from Applied Biosystems
(Inventoried primer-probes). The gene expression was normalized to
ACTB.
[0145] Semi-automated measurements of nerve fiber density in skin
biopsy. Tissue sections chosen from each biopsy were immunoassayed
with polyclonal anti-neuronal cell adhesion molecule (NCAM/CD56,
BioLegend, CA USA) antibody (1:100 dilution), using the Tyramide
Signal Amplification (TSA; Invitrogen) method for fluorescence
immunohistochemistry. Sections were analyzed and captured on Leica
DMR at 20.times. magnification. Nerve fiber density (defined as
mm/mm per section) across the entire dermal-epidermal Junction
(DEJ) was calculated by using the application Simple Neurite Tracer
on 2D images. This plugin is free software, licensed under the GNU
GPL v3 and based on the public domain image processing software
Fiji Image J. The software and step-by-step instructions are
available at http://fiji.sc/Simple_Neurite_Tracer and
http://pacific.mpi-cbg.de. Briefly, to trace a nerve fiber
(neuronal path) both the starting and end points or successive
points along the midline of a neural process were selected and
pixels generated converted to .mu.m.
[0146] Fluorescent in situ Hybridization (FISH). Fresh frozen skin
biopsies or fetal DRG were cryosectioned into 10 .mu.m slides,
fixed with chilled 10% buffered formalin (Fisher), dehydrated in
ethanol series, pretreated with protease K and hybridized using
RNAscope multiplex fluorescent assay (Advanced Cell Diagnostics,
ACD), according to the manufacturer's instruction. The probes used
were: human II17RE (ACD), human TUBB3-C2 (ACD), positive control
PPIB (ACD) and negative control DapB (ACD).
[0147] Results. Interaction of keratinocytes and nerve fibers via
IL-17c/IL-17RE during human recurrent herpes simplex virus 2
infection. There is a spatially close proximity among cutaneous
nerve endings, basal keratinocytes and CD8+ T cells in biopsy
tissues taken during HSV-2 asymptomatic reactivation (Zhu et al.,
2009; Zhu et al., 2007; Zhu et al., 2013). To explore the impact of
recurrent HSV-2 infection on peripheral nerves, the length and
width of nerve fibers positive for neuronal cell adhesion molecule
(NCAM) was measured in genital skin biopsies taken at the time of
asymptomatic reactivation and these measurements were compared to
control genital skin biopsies in contralateral sites taken from
areas without HSV reactivation. Peripheral nerve fibers in tissues
undergoing HSV asymptomatic shedding had a much higher density
compared to nerve fibers detected in control skin (FIG. 1A). Nerve
fibers in skin biopsies showing recent HSV-2 reactivation were much
longer on average, as compared to those in matching controls (n=4);
while the width of nerve fibers was similar (FIG. 1B). The increase
in nerve fiber length in genital skin biopsies during viral
asymptomatic reactivation relative to their matching control
biopsies (n=4) was 4 fold greater than those without detectable
shedding (n=8) (FIG. 1C).
[0148] NCAM+ nerve fibers in post healed skin biopsies also
co-expressed the low-affinity nerve growth factor receptor (NGFR).
Both peripherin+ and NF200+ nerve fibers were found to be present
in the papillary dermis close to the epidermis (FIG. 1D). These
results suggest that neurotrophins might be released locally to
stimulate nerve growth and/or repair nerve endings in response to
HSV-2 reactivation.
[0149] To evaluate the role(s) of keratinocytes in influencing
nerve fiber density during recurrent HSV-2 infection, individual
basal keratinocytes were selectively recovered by laser capture
microdissection (LCM) from human genital skin biopsies at the time
of acute lesion and subsequently at 4 or 8 weeks post healing as
well as contralateral control biopsies from the same patients (n=4)
and compared their transcriptional profiles (FIG. 2A). Expression
of keratin 5 and 14, markers of basal keratinocytes in the human
epidermis (Lloyd et al., 1995), was measured in isolated
keratinocytes as well as CD1a+ Langerhans cells and CD8a+ T cells
(FIG. 3) (Peng et al., 2012; Zhu et al., 2013). The isolated
keratinocytes expressed approximately 10 times higher levels of
keratin 5 and 14 than the other cell types, suggesting an enriched
cell population. Illumina Human HT-12 beadarrays contain about 300
genes annotated as growth factor/cytokine/chemokine activity; 3
were significantly induced in keratinocytes isolated from HSV-2
lesion and post healed biopsies (IL-17c, CCL5 and TNFSF10) and 6
were up-regulated only in keratinocytes from lesions (CX3CL1,
CXCL11, TNF-.alpha., CCL8, CXCL10 and CXCL9) (top panel, FIG. 2B).
Among the six related IL-17 family members, IL-17c, a predominantly
epithelial-derived cytokine (Gaffen, 2009; Ramirez-Carrozzi et al.,
2011; Song et al., 2011) was the only induced gene in keratinocytes
during recurrent HSV-2 infection (bottom panel, FIG. 2B).
Immunofluorescent staining indicated that IL-17c was expressed in a
small population of keratinocytes exclusively in the epidermis in
lesion and post healed skin biopsies with asymptomatic shedding but
not in control biopsies (FIG. 2C).
[0150] To identify the target cells of IL-17c during HSV-2
reactivation in vivo, immunofluorescent staining of IL-17RE, the
orphan receptor for IL-17c (Chang et al., 2011; Ramirez-Carrozzi et
al., 2011; Song et al., 2011), was performed. IL-17RE expression
was found not on CD15+, CD8+ or CD4+ immune cells, markers of
neutrophils and T cells, respectively, but on structures with
elongated fiber like shapes and in keratinocytes (FIG. 4A). Dual
staining for NCAM and IL-17RE and peripherin and IL-17RE showed
that IL-17RE was detected on NCAM+ and peripherin+ nerve fibers in
skin biopsies during active and asymptomatic HSV-2 infection (FIGS.
4B & 4C). To further investigate the neuronal expression of
IL-17RE, the distribution of IL-17RE protein in fetal dorsal root
ganglia (DRG) was examined. The expression of IL-17RE protein was
detected in both soma and axonal regions of DRG (FIG. 4D). Dual in
situ hybridization confirmed IL-17RE mRNA expression in
beta-tubulin 111+ neurons and also in beta-tubulin 111-cells (FIG.
4E). IL-17RE was expressed in a subset of NF200+ or peripherin+
sensory neurons (FIG. 4F), consistent with its expression patterns
observed in the genital skin (FIG. 1D). These findings indicate an
abundance of IL-17RE in the peripheral neurons and provide evidence
that IL-17c released from epidermal keratinocytes could interact
with IL-17RE on nerve fibers in the dermal area during the process
of HSV-2 reactivation.
[0151] HSV replication in human primary keratinocytes induces
IL-17c expression. To test the hypothesis that HSV-2 reactivation
could induce IL-17c in keratinocytes, human primary keratinocytes
were cultured and evaluated for whether these cells would produce
IL-17c in vitro in response to HSV infection over 12 hours. Peak
induction occurred at 6 hours post-infection (p.i) and remained at
elevated levels when infected with UV inactivated virus or
acyclovir treatment (left panel, FIG. 5A). Next, tests were
performed to determine whether HSV DNA replication activates IL-17c
expression in keratinocytes because peak IL-17c induction coincided
with early stages of HSV DNA replication, which is inhibited by
both UV and acyclovir treatment. Keratinocytes were infected with
HSV mutants containing gene deletions in the immediate-early (IE)
genes ICP0 (ICP0mu) and ICP22 (ICP22mu), or early gene ICP8
(ICP8mu), a single-stranded DNA binding protein essential for HSV
DNA replication, and the parental wild type HSV-1 strain (KOS).
Over time, cells infected with IE gene mutants had significantly
reduced levels of IL-17c. In contrast, ICP8mu induced IL-17c
expression patterned similarly as the wild type KOS strain but at
much higher levels (right panel, FIG. 5A). Immunofluorescent
staining demonstrated IL-17c expression at the cell surface and in
the cytoplasm in cultured keratinocytes infected with the HSV-2
strain HG-52 (left panel, FIG. 5B). Relative to mock infection,
HG-52 infection at MOI of 1 and 10 induced 67% and 756% more
IL-17c+ cells at 7 hours p.i, respectively (right panel, FIG. 5B).
Thus, both HSV-1 and HSV-2 induce IL1-7c expression in human
primary keratinocytes.
[0152] Peptidoglycan (PGN) and flagellin, bacterial ligands for
TLR2 and TLR5, respectively, are known to stimulate IL-17c
expression rapidly (Ramirez-Carrozzi et al., 2011). To understand
whether the signaling pathways for IL-17c induction by HSV
infection and TLR2/5 stimulation converge or are independent,
IL-17c production was evaluated after combination HSV infection and
PGN/flagellin treatment. A TLR2 neutralizing antibody blocked
PGN-dependent IL-17c expression and the combination of HSV and PGN
induced IL-17c in an additive manner (left panel, FIG. 5C). The
combination of ICP8mu infection and flagellin treatment also
additively induced IL-17c expression (right panel, FIG. 5C). These
findings show that HSV infection and PGN/flagellin independently
induce IL-17c expression in cultured human primary
keratinocytes.
[0153] To identify transcription factors that mediate IL-17c
induction, the transcriptional profiles of primary keratinocytes
were analyzed. The keratinocytes displayed high levels of
regulatory proteins involved in host innate defenses, such as
NFKB1, IRF1, IRF3, IRF7, IFI16 and PML (data not shown) (Cuchet et
al., 2011; Dev et al., 2011; Honda and Taniguchi, 2006; Orzalli et
al., 2012). Gene specific siRNA transfection reduced expression of
these transcription factors from 60 to 90% relative to control
non-specific siRNA (FIGS. 6A & 6B). siRNA knock-down of
NF-.kappa.B and IRF-3 blocked the IL-17c induction at 3 hours p.i
and reduced its induction by about 50% at 6 hours p.i, suggesting
that these two transcription factors mediate transcriptional
induction of IL-17c during the early hours of HSV infection (FIG.
5D). HSV-induced IL-17c expression was not affected by siRNA
knock-down of IRF7, IFI16 and PML, while it was significantly
inhibited by IRF1 siRNA at 3 hours p.i yet had no effect on IL-17c
induction at 6 hours p.i (FIGS. 6B & 8D).
[0154] IL-17c induces neurite growth of human neuroblastoma cells
and primary sensory neurons. To understand biological functions of
HSV induced IL-17c, the antiviral activity of IL-17c was examined.
Blocking IL-17c signaling using a neutralizing antibody for IL-17RA
or siRNA knock-down of IL-17-RE did not influence HSV gene
expression or HSV titers in human primary keratinocytes (FIG. 7).
Based on the lack of antiviral activity, the potential effect of
IL-17c on neuronal functions was explored. First, the neurotrophic
effects of exogenous IL-17c in differentiated SY5Y cells were
tested. Retinoic acid induces cell cycle arrest and differentiation
of SY5Y cells to a more neuron-like phenotype (Abemayor and Sidell,
1989). Using a two chamber microfluidic device, differentiated SY5Y
cells were found to have visible neurites extending into
microgroove channels after 24 hours in the IL-17c containing device
(FIG. 8A). The growth cone of individual neurites appears to be
larger in IL-17c containing devices (FIG. 8B). During the next 10
days, significantly more and longer neurites grew into the main
channel with basal medium plus IL-17c as compared to medium only or
medium plus NGF (FIGS. 8C & 8D). Taken together, the data show
that IL-17c can stimulate neurite growth of cultured human
neuroblastoma cells. These data were replicated using
internally-generated Hutch human IL-17c protein.
[0155] Next, human sensory neurons (HSN) were isolated from fetal
DRG. HSN were cultured in full neural medium or such medium with
IL-17c or NGF for 3 days before the cells were live imaged to
measure neurite length, branch points and cell body area hourly for
16 hours (FIGS. 9A, 10A, 10B, 10C). Neurites grew longer and faster
with more branches in the presence of IL-17c as compared to culture
medium alone or medium plus NGF; In contrast, the growth rate of
cell body were similar in all three conditions during the 16 hr
time period (FIGS. 9B & 9C). To measure the effect of IL-17c on
directional neurite growth, a 3-chamber microfluidic device was
used with HSN placed in the middle channel and the left and right
channels contained full medium (left) and full medium plus IL-17c
(right), respectively (FIG. 9D). By day 10 significantly more
neurites grew into the IL-17c containing channel than into the
medium alone channel. On day 16, cells were fixed and stained with
PGP9.5 and IL-17RE antibodies. Compared to those in the channel
with medium alone, almost twice as many neurites were found in the
IL-17c containing channel with 2.7-fold longer total neurites and
3.5-fold more branch points (FIGS. 9E & 9F). Neurites in the
IL-17c channel were IL-17RE+ and appeared to have larger growth
cones compared with those in the medium alone channel (FIGS. 9G
& 9H). Taken together, the data suggest that IL-17c might be a
neurotrophic factor promoting neurite growth and branching for
HSN.
[0156] Pre-treatment with IL-17c reduces apoptosis during HSV
infection of murine primary neurons and human primary
keratinocytes. As a previous study indicated that IL-17c provides a
survival signal for colon epithelial cells in a mouse intestinal
tumor model (Song et al., 2014), tests were performed to determine
if IL-17c has an anti-apoptotic effect on neurons. During a time
course of HSV-1 (K26) infection of mouse primary cortical neurons
(MCN), IL-17c was consistently induced throughout the time course
as compared to mock infected cells; IL-17RE was also induced (FIG.
11A). Consistent with the gene expression pattern seen in laser
captured keratinocytes in skin (FIG. 2B), IL-17a expression was not
detected by quantitative RT-PCR in MCN (data not shown). Because
the receptor for IL17c is a heterodimer of IL-17RE/IL-17RA, IL-17c
signaling was blocked using a neutralizing antibody for IL-17RA,
which had no significant effect on HSV gene expression (FIG. 12).
Next, tests were performed to determine if pre-treatment with
internally-generated Hutch exogenous murine IL-17c (mIL-17c) could
aid in survival of neurons during HSV infection. Decreased levels
of cleaved caspase 3 were detected by immunofluorescence when MCN
were pre-treated with mIL-17c, and the presence of a murine IL-17RA
neutralizing antibody (anti-mIL17RA) eliminated this reduction
(FIGS. 11B & 11C). At 16 hours p.i, there was a 29% reduction
of neurons undergoing apoptosis with mIL-17c pre-treatment before
infection (FIG. 11C). There was no significant difference of
cleaved caspase 3 profiles in neurons treated with control IgG or
anti-mIL17RA (data not shown). Consistent with these results, use
of internally-generated Hutch human IL-17c demonstrated an
anti-apoptotic effect in human keratinocytes.
[0157] Because it has already been shown that HSV infection in
human primary keratinocytes induces IL-17c and such cells have a
high level of expression of IL-17c receptors (IL-17RA and IL-17RE)
(FIGS. 5 and 7), it was next determined whether exogenous human
IL-17c (hIL17c) pre-treatment provides a pro-survival signal to K26
infected keratinocytes. Indeed, pre-treatment of keratinocytes with
hIL-17c reduced apoptosis during K26 infection and this was
reversed in the presence of a neutralizing antibody for human
IL-17RA (anti-hIL17RA) (FIG. 11D). Furthermore, blocking endogenous
IL-17c signaling with neutralizing antibodies for IL-17RA during
HSV infection of neurons and keratinocytes induced more apoptosis
(data not shown). These findings suggest that IL-17c treatment
provides a survival signal to both neurons and keratinocytes during
HSV infection.
[0158] Discussion Target derived factors such as brain-derived
neurotrophic factor (BDNF) and NGF have been described to regulate
neuronal cell function, including cell survival, axonal growth and
guidance, through retrograde signaling (Harrington and Ginty, 2013;
Tessier-Lavigne and Goodman, 1996). Here, a novel functional
interaction between mucosal keratinocytes and peripheral sensory
neurons through the IL-17c/IL-17RE pathway is described. The in
vivo data clearly demonstrate that there is peripheral nerve growth
during both clinical and subclinical HSV reactivation. The findings
that HSV infection induces IL-17c in keratinocytes, the receptor
for IL-17c is expressed on skin nerve endings located at the site
of reactivation, blocking IL-17c during HSV infection induces
neuronal apoptosis, and exogenous IL-17c induces neurite show that
this keratinocyte-peripheral nerve interaction is a the mechanism
behind the in vivo observations. One of the intriguing aspects of
the data is the demonstration that nerve growth was observed over a
prolonged 4-8 week time period post reactivation. The exact
mechanism behind this prolonged effect remains to be determined;
however in affected areas there is evidence for frequent if not
constant release of HSV-2 into the mucosal tissue, potentially
providing the stimulus for prolonged IL-17c production in
keratinocytes locally (Schiffer, 2010).
[0159] From a virus point of view, IL-17c may provide an increased
opportunity for HSV to reach more neurons for establishing latent
infection and later to gain more access to peripheral targets
during viral reactivation. Interestingly, HSV-2 glycoprotein G has
recently been proposed to regulate growth of free nerve endings in
a mouse infection model (Cabrera et al., 2015). From the host point
of view, neuron survival and growth helps preserve sensory nerve
function. This mutually beneficial interaction provides a mechanism
for the lack of hypoesthesia associated with recurrent HSV
infections and potentially explain the long standing controversy on
how HSV infection can impair peripheral nerve endings and yet not
result in any clinically discernible long standing effect on
peripheral nerve function.
[0160] Both HSV-1 and HSV-2 are neurotrophic viruses that likely
replicate in neurons before establishment of latency; little is
known about the role of neurotropic factors in HSV reactivation in
humans. The in vitro data suggest that HSV infection in HSN in vivo
would induce the expression of IL-17c, which in turn offers a
survival signal to neurons in autocrine and paracrine manners. Skin
keratinocytes also produce IL-17c during HSV infection at
peripheral sites, providing protection from apoptosis through a
similar mechanism. IL-17RA is ubiquitously expressed and it is a
shared receptor for IL-17a, IL-17c, IL-17e and IL-17f (Gaffen,
2009; Pappu et al., 2012). It is shown that the anti-apoptotic
effect of IL-17c in both keratinocytes and neurons can be
completely blocked by IL-17RA neutralizing antibodies, suggesting
that IL-17RA is required for the anti-apoptotic effect of IL-17c in
both cell types. IL-17RE expression can be detected in nerve
endings of skin biopsies during HSV-2 recurrent infection and in
both cell bodies and axons in human fetal DRGs. Taken together, it
is proposed that IL-17c from keratinocytes could bind to its
receptors (IL-17RA/IL-17RE) on nerve endings and protect axons and
cell bodies through retrograde signaling. In recent studies it was
demonstrated that IL-17c and IL-17RE and IL-17RA exist as a trimer,
providing further evidence that these in vitro observations are
operant in vivo (data not shown).
[0161] The ex vivo data clearly indicate that IL-17c can result in
both peripheral nerve growth and guidance. The molecular mechanisms
by which IL-17c promulgates neuronal growth are as yet unclear.
Nerve regeneration/repair is a tightly coordinated molecular and
cellular process that involves numerous different cell types. The
normal peripheral nerve trunk comprises complex, highly organized
structures such as the endoneurium that contain axons, Schwann
cells, macrophages, fibroblasts and blood vessels. In addition, a
mixture of inflammatory cells infiltrate to sites of nerve
injuries, adding further complexity to this microenvironment
(Cattin et al., 2015; Zochodne, 2008). In the context of recurrent
HSV-2 infection in humans, the IL-17c/IL-17RE pathway provides
crosstalk between mucosal keratinocytes and peripheral sensory
neurites that aids in nerve repair.
[0162] In summary, this Example demonstrates that keratinocytes
produce IL-17c, which stimulates nerve growth, for example during
recurrent HSV-2 infection in humans.
Example 2
[0163] Paclitaxel and vincristine have been used in the literature
for mouse studies on peripheral nerve damages and these literature
procedures will be followed (Neurobiology of Disease 2006, Melli G;
Brain research 1997, Contreras PC). Paclitaxel and vincristine will
be injected through tail vein at a concentration of 1 to 25 mg/Kg
weight.
[0164] >50 mg of murine IL-17c will be prepared for mouse
experiments. For local administration, IL-17c will be delivered by
topical cream or patch at one flank in mouse back skin. For
systematic administration, IL-17c will be delivered by
intraperitoneal injection at a concentration of 1 to 10 mg/Kg
weight.
TABLE-US-00001 TABLE 1 Experimental design for IL-17c treatment
prevention of chemotherapeutic drug induced peripheral neuropathy
in mice. Neurotrophic factor Chemotherapeutic or carrier control
drugs or vehicles Route Local or systematic Intravenous Day 1
IL-17c or PBS Day 2 IL-17c or PBS Paclitaxel or Cremophor
Vincristine Or PBS Day 3 IL-17c or PBS Day 4 IL-17c or PBS
Paclitaxel or Cremophor Vincristine Or PBS Day 5 IL-17c or PBS Day
6 IL-17c or PBS Paclitaxel or Cremophor Vincristine Or PBS Day 7
IL-17c or PBS Day 8 IL-17c or PBS Day 9 IL-17c or PBS Day 10 IL-17c
or PBS Day 11 IL-17c or PBS Day 12 IL-17c or PBS
[0165] Beneficial effects against neuropathy will be observed.
[0166] As will be understood by one of ordinary skill in the art,
each embodiment disclosed herein can comprise, consist essentially
of or consist of its particular stated element, step, ingredient or
component. Thus, the terms "include" or "including" should be
interpreted to recite: "comprise, consist of, or consist
essentially of." The transition term "comprise" or "comprises"
means includes, but is not limited to, and allows for the inclusion
of unspecified elements, steps, ingredients, or components, even in
major amounts. The transitional phrase "consisting of" excludes any
element, step, ingredient or component not specified. The
transition phrase "consisting essentially of" limits the scope of
the embodiment to the specified elements, steps, ingredients or
components and to those that do not materially affect the
embodiment. A material effect would cause a
statistically-significant reduction in the promotion of neural
growth and neural survival as measured by axon growth, axon
guidance in a particular direction, or neural cell survival in
comparison to a relevant control condition.
[0167] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention.
At the very least, and not as an attempt to limit the application
of the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques. When further clarity is required, the term
"about" has the meaning reasonably ascribed to it by a person
skilled in the art when used in conjunction with a stated numerical
value or range, i.e. denoting somewhat more or somewhat less than
the stated value or range, to within a range of .+-.20% of the
stated value; .+-.19% of the stated value; .+-.18% of the stated
value; .+-.17% of the stated value; .+-.16% of the stated value;
.+-.15% of the stated value; .+-.14% of the stated value; .+-.13%
of the stated value; .+-.12% of the stated value; .+-.11% of the
stated value; .+-.10% of the stated value; .+-.9% of the stated
value; .+-.8% of the stated value; .+-.7% of the stated value;
.+-.6% of the stated value; .+-.5% of the stated value; .+-.4% of
the stated value; .+-.3% of the stated value; .+-.2% of the stated
value; or .+-.1% of the stated value.
[0168] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing
measurements.
[0169] The terms "a," "an," "the" and similar referents used in the
context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No
language in the specification should be construed as indicating any
non-claimed element essential to the practice of the invention.
[0170] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member may be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. It is anticipated that one or more members of a group
may be included in, or deleted from, a group for reasons of
convenience and/or patentability. When any such inclusion or
deletion occurs, the specification is deemed to contain the group
as modified thus fulfilling the written description of all Markush
groups used in the appended claims.
[0171] Certain embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventor expects skilled
artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
specifically described herein. Accordingly, this invention includes
all modifications and equivalents of the subject matter recited in
the claims appended hereto as permitted by applicable law.
Moreover, any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0172] Furthermore, where references have been made to patents,
printed publications, journal articles and other written text
throughout this specification (referenced materials herein). Each
of the referenced materials are individually incorporated herein by
reference in their entirety for their referenced teaching.
[0173] In closing, it is to be understood that the embodiments of
the invention disclosed herein are illustrative of the principles
of the present invention. Other modifications that may be employed
are within the scope of the invention. Thus, by way of example, but
not of limitation, alternative configurations of the present
invention may be utilized in accordance with the teachings herein.
Accordingly, the present invention is not limited to that precisely
as shown and described.
[0174] The particulars shown herein are by way of example and for
purposes of illustrative discussion of the preferred embodiments of
the present invention only and are presented in the cause of
providing what is believed to be the most useful and readily
understood description of the principles and conceptual aspects of
various embodiments of the invention. In this regard, no attempt is
made to show structural details of the invention in more detail
than is necessary for the fundamental understanding of the
invention, the description taken with the drawings and/or examples
making apparent to those skilled in the art how the several forms
of the invention may be embodied in practice.
[0175] Definitions and explanations used in the present disclosure
are meant and intended to be controlling in any future construction
unless clearly and unambiguously modified in the following examples
or when application of the meaning renders any construction
meaningless or essentially meaningless. In cases where the
construction of the term would render it meaningless or essentially
meaningless, the definition should be taken from Webster's
Dictionary, 3rd Edition or a dictionary known to those of ordinary
skill in the art, such as the Oxford Dictionary of Biochemistry and
Molecular Biology (Ed. Anthony Smith, Oxford University Press,
Oxford, 2004).
SHORT-CITE REFERENCE LIST
[0176] Abemayor and Sidell. 1989. Environmental health perspectives
80:3-15. [0177] Cabrera, et al., 2015. Secreted herpes simplex
virus-2 glycoprotein G modifies NGF-TrkA signaling to attract free
nerve endings to the site of infection. PLoS pathogens 11:e1004571.
[0178] Cattin, et al., 2015. Cell 162:1127-1139. [0179] Chang, et
al., 2011. Immunity 35:611-621. [0180] Chuong et al., 2002.
Experimental dermatology 11:159-187. [0181] Cuchet, et al., 2011.
Journal of cell science 124:280-291. [0182] Dev, et al., 2011.
Current topics in microbiology and immunology 349:115-143. [0183]
Gaffen, 2009. Nature reviews. Immunology 9:556-567. [0184] Gaffen,
2011. Current opinion in immunology 23:613-619. [0185] Harrington
and Ginty. 2013. Nature reviews. Neuroscience 14:177-187. [0186]
Honda and Taniguchi. 2006. Nature reviews. Immunology 6:644-658.
[0187] Johnston, et al., 2013. Journal of immunology 190:2252-2262.
[0188] Johnston, et al., 2012. Lancet 379:641-647. [0189] Koutsky,
et al., 1992. The New England journal of medicine 326:1533-1539.
[0190] Lloyd, et al., 1995. The Journal of cell biology
129:1329-1344. [0191] Misery, 1997. The British journal of
dermatology 137:843-850. [0192] Orzalli, et al., 2012. Proceedings
of the National Academy of Sciences of the United States of America
109:E3008-3017. [0193] Pappu, et al., 2012. Trends in immunology
33:343-349. [0194] Peng, et al., 2009. Journal of virology
83:12559-12568. [0195] Peng, et al., 2012. Journal of virology
86:10587-10596. [0196] Ramirez-Carrozzi, et al., 2011. Nature
immunology 12:1159-1166. [0197] Roizman and Whitley, 2013. Annual
review of microbiology 67:355-374. [0198] Schiffer et al., 2010.
Proceedings of the National Academy of Sciences of the United
States of America 107:6. [0199] Schiffer, et al., 2013. Rapid
localized spread and immunologic containment define Herpes simplex
virus-2 reactivation in the human genital tract. eLife 2:e00288.
[0200] Song, et al., 2014. Immunity 40:140-152. [0201] Song, et
al., 2011. Nature immunology 12:1151-1158. [0202] Tessier-Lavigne
and Goodman, 1996. Science 274:1123-1133. [0203] Wald, et al.,
1997. J Clin Invest 99:1092-1097. [0204] Zhu, et al., 2009. Nat Med
15:886-892. [0205] Zhu, et al., 2007. The Journal of experimental
medicine 204:595-603. [0206] Zhu, et al., 2013. Nature 497:494-497.
[0207] Zochodne, 2008. Neurobiology of Peripheral Nerve
Regeneration. Cambridge University Press.
Sequence CWU 1
1
11197PRTHomo sapiens 1Met Thr Leu Leu Pro Gly Leu Leu Phe Leu Thr
Trp Leu His Thr Cys 1 5 10 15 Leu Ala His His Asp Pro Ser Leu Arg
Gly His Pro His Ser His Gly 20 25 30 Thr Pro His Cys Tyr Ser Ala
Glu Glu Leu Pro Leu Gly Gln Ala Pro 35 40 45 Pro His Leu Leu Ala
Arg Gly Ala Lys Trp Gly Gln Ala Leu Pro Val 50 55 60 Ala Leu Val
Ser Ser Leu Glu Ala Ala Ser His Arg Gly Arg His Glu 65 70 75 80 Arg
Pro Ser Ala Thr Thr Gln Cys Pro Val Leu Arg Pro Glu Glu Val 85 90
95 Leu Glu Ala Asp Thr His Gln Arg Ser Ile Ser Pro Trp Arg Tyr Arg
100 105 110 Val Asp Thr Asp Glu Asp Arg Tyr Pro Gln Lys Leu Ala Phe
Ala Glu 115 120 125 Cys Leu Cys Arg Gly Cys Ile Asp Ala Arg Thr Gly
Arg Glu Thr Ala 130 135 140 Ala Leu Asn Ser Val Arg Leu Leu Gln Ser
Leu Leu Val Leu Arg Arg 145 150 155 160 Arg Pro Cys Ser Arg Asp Gly
Ser Gly Leu Pro Thr Pro Gly Ala Phe 165 170 175 Ala Phe His Thr Glu
Phe Ile His Val Pro Val Gly Cys Thr Cys Val 180 185 190 Leu Pro Arg
Ser Val 195
* * * * *
References